Method for use of microdialysis

ABSTRACT

Very accurate measurements of mass transfer can be made rapidly by permitting diffusion of an agent desired to be measured into or out of a small, very precisely known volume of a microdialysis probe, then rapidly pumping or flushing (“pulsing”) the probe with a known volume of fluid as a single pulse. The diffusion and pulsing may be repeated. The method, hereinafter called pulsatile microdialysis (PMD) to distinguish it from prior art continuous flow microdialysis, is useful for measurements in a number of processes, including protein binding, adsorption to binding agents such as activated charcoal, release from microemulsion drug delivery systems, determination of drug diffusion coefficients and concentrations, and for various other purposes. 
     The method is based on mathematical manipulation of Fick&#39;s Laws. Resulting equations were verified against experimental data using methazolamide, warfarin and benzocaine as test drugs.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to an improved method of effecting and measuringmass transfer. In particular, the invention relates to the use of animproved method of microdialysis for measuring the transfer ofrelatively small quantities of dissolved, suspended or otherwisedispersed material between two media (one inside and one outside themicrodialysis probe). The transfer can be characterized by the loss ofmaterial from the medium in which it is contained and/or the collectionby the other medium, and in particular can be used to sample drugconcentrations and/or characterize the rates at which various processesoccur and the extent of transfer. Examples include determination of drugsolubility, and processes such as binding of drugs to proteins,chelation and complexation of drugs, adsorption of drugs in solutiononto charcoal and other adsorbing agents, and release of drugs fromemulsions and microemulsion systems. In addition, the rate of transfercan be used to determine the diffusion coefficients of drugs andpermeabilities of coatings placed on the probe window. (Although theseexamples involve drugs and are of pharmaceutical interest, the inventionextends to any chemical, particle or droplet that can transfer betweentwo media by passing through a membrane). Other applications includedetermining drug dissolution rates, and precipitation/crystallizationrates of a dissolved drug from supersaturated solutions. Otherapplications within the scope and intent of the invention will occur tothose skilled in the art.

2. Summary of the Prior Art

Microdialysis performed in a continuous manner is a known method forsampling drug concentrations from media in biological tissues or invitro systems; however, certain deficiencies, as will be discussed morefully below, have prevented its optimum application. The prior arttechnique is based on the dialysis principle, employing a“semipermeable” membrane, i.e., one that is highly permeable to waterand small molecules. In this method, a sampling solution (dialysate) isperfused continuously through a probe, and a drug or other material ofinterest passively diffuses into the dialysate from the surroundingmedium. The dialysate is collected and analyzed for drug content, andthe concentration of drug or other material of interest in thesurrounding medium is then estimated from that information. (Ananalogous procedure can be done in which the dialysate is the donor, andthe amount of drug lost to the surrounding medium is determined. This isoften referred to as retrodialysis or retromicrodialysis.)

Microdialysis can offer significant advantages compared to othersampling methods. For instance, since microdialysis probes are verysmall, they can be placed directly into biological tissue for in vivotesting or into small “receivers” for in vitro systems. In addition, themethod offers the advantage of a clean aqueous sample withoutpre-detection sample preparation, such as separation or clean up steps.Consequently, microdialysis is becoming a standard technique for in vivoand in vitro analysis of drug and biochemical concentrations.

In the standard microdialysis method, dialysate is continuously perfusedthrough the probe, usually at a constant flow rate. (This will bereferred to as continuous flow microdialysis, or CFMD.) For purposes ofthis invention, the membrane will be referred to as highly permeable,i.e., it is permeable to water and relatively small molecules, particlesand droplets (e.g., from a microemulsion) but impermeable to relativelylarge molecules such as proteins, etc. The essential parameter, ofcourse, is that the membrane be permeable with respect to the material,e.g., a drug, that is to be measured or withdrawn by means of diffusion.The choice of perfusion flow rate for the dialysate is governedprimarily by the sample size for the analysis. Typical CFMD perfusionflow rates range from 0.5 to 2.0 μL/min for samples that will beanalyzed by high-pressure liquid chromatography (HPLC) methods, forexample. At these flow rates, however, the time required for sampling isrelatively long, and the time resolution of the samples (i.e., theability to associate a specific concentration with a specific time or ashort time interval) is poor. In addition, there are problems associatedwith generating sufficient sample volumes (5-20 μL) in short timeintervals (less than 30 seconds, perhaps less than 5-10 seconds). Forinstance, the sample concentrations become very dilute and may fallbelow the detection limit of the assay being utilized. Consequently,CFMD is poorly suited for studies in which concentrations changerelatively rapidly. Such cases arise frequently in pharmacy and biology,and can include in vitro cellular drug uptake kinetics studies orbinding studies, drug complexation, drug adsorption to charcoal or otherbinding agents, precipitation from supersaturated solutions, etc. Forexample, it has been reported that methazolamide uptake by red bloodcells suspended in buffer is very rapid at early times, with the bufferconcentration decreasing by 50% in the first 1-2 minutes. For othersystems, such as protein binding, a 50% decrease in concentration mayoccur in less than 10-15 seconds. For setups like these, the inabilityof CFMD to sample every 10-15 seconds is a great disadvantage. Inaddition, for sampling methods such as spiking, which requiresseparating the cells from the buffer, large errors can potentially occurbecause the uptake process continues during the sample preparation.Thus, a microdialysis method that can offer good time resolution withinrelatively short time frames would offer significant advantages forsystems like these.

Another problem that can be associated with CFMD is that, at typicalperfusion flow rates, the recovery of drug and the resulting samplingefficiency can be poor. The recovery of a drug is the relationshipbetween concentrations of the drug in the donor fluid and that of thedialysate, and the fraction recovered (F_(R)) is defined in terms of theratio of the dialysate concentration (C_(S)) and donor concentration(C_(D)). For dialysate initially void of drug, and when C_(D) can betaken as constant, this is given as

$\begin{matrix}{F_{R} = \frac{C_{S}}{C_{D}}} & (1)\end{matrix}$In vitro, a number of parameters influence the F_(R), including thetemperature, flow rate, probe length, and the physical properties of thedrug, perfusate and membrane. Since the perfusion is continuous in CFMD,equilibrium between the dialysate and the donor medium is notapproached, and the F_(R) is typically low.

For retrodialysis, the analogous parameter would be the fractionremaining in the dialysate, R_(F). Denoting the concentration in thedialysate before entering the probe as C₀, this is defined as

$\begin{matrix}{R_{F} = \frac{C_{S}}{C_{0}}} & (2)\end{matrix}$

For situations in which the concentration of the external medium changesappreciably during the time a microdialysis sample is taken, the F_(R)defined above is not applicable because C_(D) is changing with time.Thus, a method for determining the C_(D) at specific times is needed. Asdiscussed above, this is further complicated by the fact that takingsamples rapidly is often difficult because processes can be ongoingduring separation or other cleanup steps prior to sample assay. Thus,the need for a method to obtain specific values of C₀ at specific timesusing a fast method is apparent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a microdialysis probe.

FIG. 2 is a graph of the fraction of material recovered at various flowrates for pulsatile microdialysis vs. continuous flow microdialysis inwhich the donor is the medium outside the probe and does not change withtime.

FIG. 3 shows calibration curves for two different probes for the case inwhich the donor medium is outside the probe and does not change withtime.

FIG. 4 shows calibration curves for two different resting times for thesame probe, for the case in which the donor medium is outside the probeand does not change with time.

FIG. 5 is a curve showing a measure of fraction recovered (F_(R)) as afunction of the exposure time, in which the donor medium is outside theprobe and does not change with time.

FIG. 6 is a curve showing a measure of In (1−F_(RP)) as a function ofthe exposure time.

FIG. 7 is a curve showing the fraction recovered (F_(R)) as a functionof 1/V_(S).

FIG. 8 is a curve showing the log of the concentration as a function oftime for first-order drug uptake for a case in which the drugconcentration in the donor changes slowly and the donor is the mediumoutside the probe.

FIG. 9 is a curve showing the concentrations in the sample and donor asa function of time for a rapid first order decline in the drugconcentration, in which the donor is outside the probe.

FIG. 10 is a curve showing the log of the concentration as a function oftime for a rapid first order decline in the drug concentration, in whichthe donor is outside the probe.

FIG. 11 is a curve comparing the result of pulsatile microdialysis anddirect donor sampling for the adsorption of methazolamide to activatedcharcoal.

FIG. 12 is a curve showing the free concentration of warfarin as afunction of time in the presence of bovine serum albumin.

FIG. 13 is a curve showing the release of benzocaine from amicroemulsion system as a function of time.

SUMMARY OF THE INVENTION

The invention comprises a method for transferring (withdrawing ordonating) by diffusion at least some of a diffusible material(hereinafter variously called an agent or drug) contained (eitherdissolved or suspended) in a medium either inside or outside of amicrodialysis probe.

In its broadest sense, the invention relates to pumping a dialysate in apulsed manner and analyzing at least some of such dialysate for itscontent of a desired material. More specifically, the invention relatesto a microdialysis process comprising pumping a dialysate through aprobe, with an improvement comprising pumping the dialysate in a pulsedmanner and analyzing at least some of such dialysate for its content ofa desired material.

Still more specifically, the invention provides a method of performingmicrodialysis comprising:

-   -   a) providing a microdialysis probe comprising a section of        relatively highly permeable membrane connected at its inlet to a        source of dialysate and at its outlet to a receiver, and through        which membrane a diffusible agent is to be transferred;    -   b) putting said probe in contact with said medium from which the        at least some of said contained material is to be withdrawn, or        to which some of the said material is to be donated, by        diffusion;    -   c) perfusing a known quantity of a dialysate, which may or may        not contain said agent, into the highly permeable section of the        probe;    -   d) allowing said known quantity of dialysate to remain        stationary for a period of time, called a resting time,        sufficient to permit at least some of said contained material to        diffuse into the dialysate from the external medium (acting as a        donor medium), or to diffuse from the dialysate into the        external medium (acting as a receiver medium);    -   e) flushing out (“pulsing”) said known quantity of dialysate at        a high flow rate with a single pulse of known volume of        dialysate into a receiving container for subsequent analysis.

Because of the pulsing feature and the way it is applied in the presentinvention, which is believed to be unique to the present invention, themethod is referred to herein as pulsatile microdialysis.

Pulsatile microdialysis has surprisingly been found to provide not onlyaccurate but very rapid results, thus greatly facilitating, forinstance, the ability to correlate in vitro with in vivo results in thecase of drugs. Similarly, the methods of this invention havesurprisingly been found applicable to determining a free drugconcentration, as well as the concentration of the free form in thepresence of other forms, including bound, precipitated, adsorbed, orcomplexed forms, or the presence of proteins, enzymes, and other largemolecules, structures or particles. In addition, the present inventionhas surprisingly been found to be very useful for accurately determiningthe permeability of a porous device such as a microdialysis probe, andfor accurately determining other properties of such a probe such as itswindow volume. Moreover, the instant invention provides surprisinglyaccurate measurement of diffusion coefficients, the rate of release ofan agent dissolved in emulsion and/or microemulsion droplets, the rateof binding of a material under conditions of adsorption to particles,complexation, and chemical reaction.

For all the above uses, the present invention has been found to beextremely valuable for the accurate determination of changes in quicklychanging systems.

In a preferred embodiment, the invention comprises the method describedabove wherein said probe comprises a tubular section of highly permeablemembrane connected at each end to a section of relatively impermeablemembrane or section of the probe.

In another preferred embodiment, the invention comprises a method asdescribed above wherein said probe comprises a needle-type probecomprising a highly permeable tubular membrane concentrically positionedwith a relatively impermeable tube. It will be understood by thoseskilled in the art that such terms as “highly” and “relatively,” and thelike, do not connote specific numerical values. They are to beinterpreted in the context of the materials involved in both theconstruction of the probe and in the context of the materials (agents)desired to be diffused into the probe or left behind, as the case maybe. For example, in the case of a highly diffusible agent, the“relatively” impermeable membrane may need to be essentiallyimpermeable, e.g., glass. On the other hand, in the case of an agent oflow diffusibility such as a large molecule or an agent bound to anothermaterial, the “relatively” impermeable area may have significantpermeability to various substances but yet be sufficiently impermeableto the agent to be separated so as to be satisfactorily operable in themethods of the invention. Accordingly, the term “relatively” is intendedto permit a reasonably broad choice of materials of construction for theprobe.

In still another preferred embodiment, the invention comprises a methodas described above wherein said probe comprises a well with an inlet andoutlet that is separated from another well by a highly permeablemembrane.

In yet another preferred embodiment, the invention comprises a method asdescribed above wherein said probe, of either type mentioned in theprevious two paragraphs, is directly connected to an apparatus forassay, such as direct connection to liquid chromatography equipment(HPLC).

As used herein, the term “probe” is intended to include any device ofany geometry which is capable of being used in the methods described inthe claims.

It will be readily understood by those skilled in the art that the terms“agent” and “drug” are often used interchangeably, and it will befurther understood that the term “agent” is the broader term and isintended to include not only drugs but also any other pharmaceuticallyor chemically active material that will function in the presentinvention for any purpose for which the methods and mathematics of thepresent invention may be applied.

It will also be readily understood that while the instant invention isoften described herein in connection with specific mathematicalexpressions, the spirit and advantages of the invention may also bepossibly realized by application of other mathematical not disclosedherein but which do not avoid or limit the fundamental nature and scopeof the invention.

DISCUSSION OF THE INVENTION

The approach taken by the instant invention to solving the problemsassociated with the conventional microdialysis method, referred to ascontinuous flow microdialysis (CFMD), is the use of a novel method ofmicrodialysis, referred to as pulsatile microdialysis (PMD). In thismethod, the dialysate is pumped into the probe and then allowed toremain at rest for a brief, discrete period referred to as the restingtime (t_(R)). After a suitable t_(R) (typically 3-100 seconds,preferably 3-15 seconds), the dialysate is flushed (i.e., pumped) outand collected for assay. It is usually preferred that this flushing isdone as a single pulse at a relatively high flow rate (typically 50-165μL/min), preferably to minimize or eliminate the effects of furtherdiffusion, which usually simplifies mathematical analysis of the data.The PMD method allows flexibility to optimize the experimentalprocedure. For instance, the t_(R) can be chosen to be long enough togive an acceptable amount of transfer between the dialysate and externalmedium by diffusion, and short enough to provide the desired timeresolution. In addition, the volume that is flushed (i.e., the samplevolume) is chosen to completely collect the sample that was at rest inthe probe window while minimizing its dilution. (For determining theconcentration in the external medium, the time must be chosen longenough to allow enough of the drug to be collected by the dialysate,which is characterized by the fraction recovered F_(R), which wasdefined by Equation (1). By analogy, when the dialysate is being used asthe donor medium, the time must be long enough so some fraction of thedrug in the dialysate is lost to the external medium, as given byEquation (2) for R_(F)). As a result, the PMD method offers twoadvantages over CFMD: First, the pulsatile method shows excellent timeresolution, even for rapidly changing concentrations; second, the F_(R)can be made higher with PMD by increasing the t_(R), which can enablethe detection of low concentrations of drug in the surrounding fluidsusing shorter time intervals than would be possible using CFMD.

GLOSSARY OF TERMS a inner radius of the microdialysis probe window Aarea of the probe window = 2πaL β_(n) roots of Equation (8) CFMDcontinuous flow microdialysis C_(D) concentration in the donor solutionC₀ concentration in dialysate before entering probe C_(S) averageconcentration in a collected dialysate sample (=M/V_(S)) D diffusioncoefficient of the drug in the dialysate δ_(n) defined by Equation (18)F_(R) fractional recovery for a sample, defined by Equation (1) F_(R) ⁰PMD concentration divided by concentration in donor at start of sampleinterval, defined by Equation (75) F_(RQ) fractional recovery of thecontinuous portion of the PMD sample F_(RP) fractional recovery of thepulsed portion of the PMD sample γ_(n) defined by Equation (17) hthickness of the wall of the probe window (outer minus inner radius) Llength of the microdialysis probe (=V_(W)/πa²) λ defined by Equation (9)M total amount of drug in the collected a dialysate sample M_(Q) amountof drug in the sample portion that did not rest in the probe windowM_(P) amount of drug in the sample portion that rested in the probewindow P permeability of the probe window PMD pulsatile microdialysis Qflow rate (μL/min) R_(F) The fraction remaining in the dialysate (whenthe dialysate is the donor), defined by Equation (2) R_(FQ) fractionremaining in the dialysate for the continuous portion of the PMD sampleR_(FP) fractional remaining in the dialysate for the pulsed portion ofthe PMD sample t_(P) exposure time for the pulsed portion of thedialysate sample (t_(R) + t_(Q)) t_(Q) transit time for the continuousportion of the dialysate sample = V_(W)/Q t_(R) resting tune fordialysate in the probe window t_(S) duration of sampling time interval =V_(S)/Q τ tortuosity of pores in the probe window wall V_(W) probewindow volume = πa²L (same as the volume of dialysate allowed to rest)V_(S) volume of one dialysate samplePulsatile Microdialysis: Mathematical Model for a Constant Concentrationin the Medium Outside the Probe

A prototype microdialysis setup is shown in FIG. 1, which illustrates aprobe window made of a highly permeable tube of constant inner radius a,length L and volume V_(W). In the most general case, microdialysis canbe described in cylindrical coordinates as a transport of drug thatoccurs by a combination of passive diffusion in the radial direction,and convection plus passive diffusion in the axial direction. This iswritten mathematically as

$\begin{matrix}{\frac{\partial C}{\partial t} = {{{- v_{z}}\frac{\partial C}{\partial z}} + {D\frac{\partial^{2}C}{\partial z^{2}}} + {\frac{D}{r}\frac{\partial}{\partial r}\left( {r\frac{\partial C}{\partial r}} \right)}}} & (3)\end{matrix}$

Here, C is the concentration of the dialysate inside the probe at agiven position and time, D is the diffusion coefficient of the drug inthe dialysate, and v_(z) is the axial velocity, which in general is afunction of r but is typically (i.e., for CFMD) held constant withrespect to time. On the right hand side of Equation (3), the first termrepresents the effects of convection, while the second and third termsrepresent the contribution the axial and radial diffusion, respectively.For the case of PMD, Equation (3) can be simplified as follows:

-   -   While the dialysate is stationary in the probe, v_(z)=0 and the        convection term can be ignored.    -   The dialysate is moved into and out of the probe window quickly        and completely. Thus, the exposure time (the time spent inside        the probe window, and where diffusion can occur into or out of        the probe) for any portion of the dialysate sample is well        defined.    -   The exposure time of the sample is chosen to be short enough to        neglect axial diffusion. From the theory of separation of        variables, for a tube of radius a, the relaxation time        characteristic of the approach to equilibrium for diffusion in        the radial direction is ˜a²/D (Carslaw and Jaeger, Conduction of        Heat in Solids, Clarendon Press, Oxford, 1985). From random walk        theory, the average distance traveled by diffusing molecules        during a time interval t is ˜√{square root over (Dt)} (Reichl, A        Modern Course in Statistical Physics, U. Texas Press, Austin,        1980, Chapter 6.). When the exposure time is comparable to the        relaxation time, the average axial distance traveled due to        diffusion is ˜a. Since a<<L for microdialysis probes, axial        diffusion will have a negligible effect on the mass balance in        the sample.    -   Even when the dialysate is being flushed, the exposure time for        the flowing sample is short enough so the axial gradient does        not have time to develop, and the

$v_{z}\frac{\partial C}{\partial z}$may be neglected.As a result, Equation (3) reduces to

$\begin{matrix}{\frac{\partial C}{\partial t} = {\frac{D}{r}\frac{\partial\;}{\partial r}\left( {r\frac{\partial C}{\partial r}} \right)}} & (4)\end{matrix}$Equation (4) is a partial differential equation that requires oneinitial condition and two boundary conditions for its complete solution.The initial condition is that the dialysate is initially void of drug(or other material to be separated) when it enters the probe region ofthe microdialysis tube. The boundary conditions are, in part, obtainedfrom the following considerations:

-   -   The concentration in the medium outside the probe is constant        (or may be approximated as constant) during each sampling        period.    -   The drug concentration is finite everywhere in the microdialysis        probe.    -   The dialysis tube wall is very thin and highly permeable, so        pseudo-steady-state in the wall is established quickly. Thus,        the flux of drug from the donor into the dialysate is        proportional to the concentration difference across the wall of        the probe window. The proportionality factor is the permeability        P of the probe window, which is assumed to remain constant. The        permeability is defined by the equation

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t} = {{AP}\left( {C_{D} - C_{R}} \right)}} & (5)\end{matrix}$

-   -   where dM/dt is the rate at which the drug crosses the probe        window wall, A is the area of the probe window, and C_(D)-C_(R)        is the concentration difference across the membrane (i.e., the        difference in concentrations in the two liquid media at the        inner and outer surfaces of the membrane).

Two cases will be considered below. The first is the case in which thedonor is the medium outside the probe and the dialysate accumulates drugfrom the donor. The second is the case in which the dialysate is thedonor and loses drug to the medium surrounding the probe.

The Donor Medium is Outside the Probe

When the medium outside the probe is the donor, then C_(D) correspondsto the concentration outside the probe and C_(R) is the concentration inthe dialysate near r=a. For this case, the boundary and initialconditions are mathematically written as

$\begin{matrix}\begin{matrix}{{Initial}\mspace{14mu}{condition}} & {{C\left( {r,0} \right)} = 0} & {t = 0} \\{{Boundary}\mspace{14mu}{conditions}} & {{C\left( {0,t} \right)} = {finite}} & {r = 0} \\\; & {{{- D}\frac{\partial C}{\partial r}} = {P\left( {C_{D} - C} \right)}} & {r = a}\end{matrix} & (6)\end{matrix}$Using the separation of variables method, Equations (4) and (6) can besolved to give the concentration in a volume element of dialysate at agiven radius as

$\begin{matrix}{{C\left( {r,t} \right)} = {C_{D}\left\lbrack {1 - {2{\sum\limits_{n = 1}^{\infty}{\frac{\lambda\;{J_{0}\left( {\beta_{n}{r/a}} \right)}}{\left( {\beta_{n}^{2} + \lambda^{2}} \right){J_{0}\left( \beta_{n} \right)}}{\exp\left( {- \frac{\beta_{n}^{2}{Dt}}{a^{2}}} \right)}}}}} \right\rbrack}} & (7)\end{matrix}$Here, t is the length of time that a given volume element of dialysatewas in the probe window (the exposure time), J₀ and J₁ are thezero-order and first-order Bessel function of the first kind,respectively (Carslaw and Jaeger, op. cit.; Ozisik, Boundary ValueProblems of Heat Conduction, Dover Publications, New York, 1989), andthe β_(n) are the roots of the equationβ_(n) J ₁(β_(n))−λJ ₀(β_(n))=0  (8)where

$\begin{matrix}{\lambda = \frac{aP}{D}} & (9)\end{matrix}$Values of β_(n) have been tabulated for various values of and n in theliterature (Crank, The Mathematics of Diffusion, Clarendon Press,Oxford, 1975), and can also be calculated from Equation (8) using thenonlinear solvers included with spreadsheets such as EXCEL®. For theprobes used here, it can be assumed that the drug does not partitioninto the probe material, and thus permeates the probe window wallexclusively through pores. If the donor and receiver media are similar,the partition coefficients between the pore medium and the donor ordialysate may be taken as unity. Thus, denoting the probe windowporosity, thickness (difference between the outer and inner radii) andtortuosity by ε, h and τ, respectively, the permeability of the windowis given by

$\begin{matrix}{P = \frac{ɛ\; D}{\tau\; h}} & (10)\end{matrix}$which can be combined with Equation (9) to give

$\begin{matrix}{\lambda = \frac{a\; ɛ}{\tau\; h}} & (11)\end{matrix}$Thus, for these probes, λ, depends on properties of the probe window,but not properties of the drug or solvent. However, this is a specialcase of Equation (9). If the probes are coated or modified, then λdepends not only on the geometrical properties of the probe, but alsothe material interactions between the drug and probe, and Equation (9)must be used.

The total amount of drug collected by the dialysate in the probe windowafter a given exposure time is found by integrating the concentrationover the volume of the sample. Since the axial dependence is neglectedin the mass balance, the mass in a sample of volume V with an exposuretime t can be found from

$\begin{matrix}{M = {\frac{V}{\pi\; a^{2}}{\int_{0}^{a}{2\pi\;{{rC}\left( {r,t} \right)}\ {\mathbb{d}r}}}}} & (12)\end{matrix}$

Not all parts of a collected dialysate sample will be exposed to thedonor for the same length of time, and two portions must be considered.One portion of the sample (referred to as the continuous portion) flowsthrough the probe window without resting. The other portion (referred toas the pulsed portion) is pumped into the window, allowed to remain atrest for a resting time t_(R), and then pumped out. For the continuousportion, the dialysate exposure time is simply the transit time t_(Q)required for an element of fluid to move through the probe window. Theexposure time t_(P) for the pulsed portion is the sum of the resting andtransit times. These are given, respectively, by

$\begin{matrix}{t_{Q} = {{\frac{V_{W}}{Q}\mspace{14mu}{and}\mspace{14mu} t_{P}} = {t_{R} + t_{Q}}}} & (13)\end{matrix}$where Q is the flow rate (volume per time) of the flushing. Thus, for asample of volume V_(S), the pulsed portion has a volume V_(W) andaccumulates a mass Mp during a total exposure time of t_(P), while thecontinuous portion has a volume V_(S)−V_(W) and accumulates a mass M_(Q)during an exposure time of t_(Q). M_(p) can be found by setting t=t_(P)in Equation (7), performing the integration in Equation (12), andmultiplying by the length of the probe window V_(W)/ πa². M_(Q) can befound by setting t=t_(Q) in Equation (7), performing the integration inEquation (12), and multiplying by a length (V_(S)−V_(W))/πa². The totalmass of drug Min a collected sample is given byM=M _(P) +M _(Q)  (14)where

$\begin{matrix}{M_{P} = {V_{W}{C_{D}\left\lbrack {1 - {\sum\limits_{n = 1}^{\infty}{\delta_{n}{\exp\left( {{- \gamma_{n}}t_{P}} \right)}}}} \right\rbrack}}} & (15) \\{M_{Q} = {\left( {V_{S} - V_{W}} \right){C_{D}\left\lbrack {1 - {\sum\limits_{n = 1}^{\infty}{\delta_{n}{\exp\left( {{- \gamma_{n}}t_{Q}} \right)}}}} \right\rbrack}}} & (16)\end{matrix}$The constants γ_(n) are δ_(n) are given by

$\begin{matrix}{\gamma_{n} = \frac{\beta_{n}^{2}D}{a^{2}}} & (17) \\{\delta_{n} = \frac{4\lambda^{2}}{\beta_{n}^{2}\left( {\beta_{n}^{2} + \lambda^{2}} \right)}} & (18) \\{where} & \; \\{{\sum\limits_{n = 1}^{\infty}\delta_{n}} = 1} & (19)\end{matrix}$(It should be noted that this form of γ_(n) holds only for dialysatesolutions. For two-phase systems, such as microemulsions, the form ofγ_(n) may change, but the use of γ in the subsequent equations isexpected to remain the same.)

When the concentration in the external medium is constant, or can beapproximated as constant, the fractional recovery in the sample, definedin Equation (1), can be expressed in terms of the mass in the sample andthe sample volume asF _(R) =M/V _(S) C _(D M=V) _(S) C _(S)  (20)Similarly, the fractional recoveries of the pulsed (F_(RP)) andcontinuous (F_(RQ)) portions of the sample are defined as

$\begin{matrix}{F_{RP} = {\frac{M_{P}}{V_{W}C_{D}} = {1 - {\sum\limits_{n = 1}^{\infty}{\delta_{n}{\exp\left( {{- \gamma_{n}}t_{P}} \right)}}}}}} & (21) \\{F_{RQ} = {\frac{M_{Q}}{\left( {V_{S} - V_{W}} \right)C_{D}} = {1 - {\sum\limits_{n = 1}^{\infty}{\delta_{n}{\exp\left( {{- \gamma_{n}}t_{Q}} \right)}}}}}} & (22)\end{matrix}$The total mass in the sample can be written asM=V _(W) C _(D) F _(RP)+(V _(S) −V _(W))C _(D) F _(RQ)  (23)Since M is proportional to the donor concentration C_(D), linearcalibration plots relating C_(D) and C_(S) can be constructed accordingto Equation (1). It is also possible to rewrite Equation (23) as

$\begin{matrix}{F_{R} = {{\frac{V_{W}}{V_{S}}\left( {F_{RP} - F_{RQ}} \right)} + F_{RQ}}} & (24)\end{matrix}$For a constant flow rate, F_(RQ) corresponds to the fractional recoveryfor CFMD, as can be seen from Equations (21), (22) and (24) whent_(P)=t_(Q), (equivalently, t_(R)=0). This provides a procedure forobtaining the F_(RQ) from F_(R) data according to the condition that

$\begin{matrix}{F_{RQ} = {\lim\limits_{t_{R}\rightarrow 0}F_{R}}} & (25)\end{matrix}$

This can be done in practice by fitting the F_(R) vs. t_(R) to anempirical function using nonlinear regression, and then taking the valueof the function for t_(R)=0. Since the above theory predicts that F_(R)varies exponentially with the exposure time, this is using the equationsbelow:F _(R) =a ₁ exp(−b ₁ t _(R))+a ₂ exp(−b ₂ t _(R))+a ₃  (26)F _(RQ) =a ₁ +a ₂ +a ₃  (27)

Since F_(R) and F_(RQ) can be determined experimentally (see Equation(1) and Example 1, respectively), it is possible to obtain F_(RP) forany exposure time t_(P) from Equation (24). However, this requiresaccurately knowing the window volume V_(W). In practice, when a probe isfirst used, it is calibrated to determine V_(W), which then allows theF_(RP) to be calculated in any subsequent experiment using that probe.(It should be noted that simply calculated V_(W) using manufacturer'sspecifications or optical measurements is not accurate enough for manyof the analyses presented here. Thus, it is preferred to measure V_(W)by plotting F_(R) vs. 1 /V_(S), using a constant resting time with aknown value of F_(RP). This discussed more fully below.)

The approach to equilibrium is characterized in the above equations bythe exponential transient terms in the infinite series. For all valuesof λ and n, both the δ_(n) and exponential terms are between zero andone, and both tend toward zero with increasing n or time of exposure.Using typical values for the dialysis probes used in this study(a˜100−150 μ, h=8−12 μ, ε<0.05) and a typical tortuosity value(τ>1.5-2), Equation (11) shows that 0<λ<0.5. For this range of λ,numerical calculations show that δ₁>0.99 and δ₂/δ₁<0.005. Thus, there isnegligible error (less than 0.1-0.5%) introduced by neglecting the n>1terms, so Equations (21) and (22) can be written as

$\begin{matrix}{F_{RP} = {\frac{M_{P}}{V_{W}C_{D}} = {1 - {\delta_{1}{\exp\left( {{- \gamma_{1}}t_{P}} \right)}}}}} & (28) \\{F_{RQ} = {\frac{M_{Q}}{\left( {V_{S} - V_{W}} \right)C_{D}} = {1 - {\delta_{1}{\exp\left( {{- \gamma_{1}}t_{Q}} \right)}}}}} & (29) \\{{\ln\left( {1 - F_{RP}} \right)} = {{\ln\;\delta_{1}} - {\gamma_{1}t_{P}}}} & (30)\end{matrix}$where

$\begin{matrix}{\gamma_{1} = {\frac{\beta_{1}^{2}D}{a^{2}} = \frac{\pi\; L\;\beta_{1}^{2}D}{V_{W}}}} & (31) \\{\delta_{1} = \frac{4\lambda^{2}}{\beta_{1}^{2}\left( {\beta_{1}^{2} + \lambda^{2}} \right)}} & (32)\end{matrix}$whereβ₁ J ₁(β1)−λJ ₀(β₁)=0  (33)and F_(RP) is determined from Equation (24).

In theory, plots of ln(1−F_(RP)) vs. t_(P) can be used to determine γ₁and δ₁ (which, in turn, can be used in Equations (8) and (18) to findλ). This is true for finding γ₁ because small experimental errors willminimally affect the slope. However, the intercept is typically close tozero because δ₁ is close to 1, so experimental errors can result insignificant relative errors in the intercept. Because small errors inthe value of δ₁ can result in relatively large errors in thecorresponding λ, the approximations are made thatδ₁=1 neglect n>1 terms  (34)F _(RP)=1−exp(−γ₁ t _(P))  (35)F _(RP)=1−exp(−γ₁ t _(Q))  (36)Since δ₁ is taken as 1, only γ₁ is obtained from a plot of Equation(35). However, to accurately find the F_(RP) and avoid possiblysubstantial errors in value of γ₁, it is necessary that the windowvolume V_(W) be accurately known. (Methods to determine V_(W) arepresented below.) It is also possible to obtain γ₁ using an alternativemethod, which is given by Equation (47) below.

The probe window wall permeability coefficient can be calculated usingPMD as well. From Equations (13), (28) and (34), the mass in the pulsedportion of the sample is given byM _(P) =V _(W) C _(D) F _(RP) =V _(W) C _(D)[1−exp(−γ₁ t _(R))]Using Equation (14) and noting that M_(Q) is constant when all samplesare taken in the same manner, the rate of uptake of the drug into thedialysate is given by

$\frac{\mathbb{d}M}{\mathbb{d}t} = {\frac{\mathbb{d}M}{\mathbb{d}t_{r}} = {V_{W}C_{D}\gamma_{1}{\exp\left( {{- \gamma_{1}}t_{Q}} \right)}{\exp\left( {{- \gamma_{1}}t_{R}} \right)}}}$Using Equation (29) gives

$\frac{\mathbb{d}M}{\mathbb{d}t} = {V_{W}C_{D}{\gamma_{1}\left( {1 - F_{RQ}} \right)}{\exp\left( {{- \gamma_{1}}t_{R}} \right)}}$If t_(R)=0, this can be written as

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t} = {V_{W}C_{D}{\gamma_{1}\left( {1 - F_{RQ}} \right)}}} & (37)\end{matrix}$At very early times (t_(R) approaching zero), the receiver concentrationC_(R) (here, corresponding to the dialysate concentration near the probemembrane) is negligible compared to the donor concentration C_(D)(corresponding here to the concentration in the external solution), andEquation (5) can be simplified to

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t} = {APC}_{D}} & (38)\end{matrix}$Here, A is the area of the probe window, which can be obtained fromV_(W) and the length of the window L (which is easily measured).Combining Equations (37) and (38) leads to

$P = \frac{V_{W}{\gamma_{1}\left( {1 - F_{RQ}} \right)}}{A}$

A refinement can be done as follows. The value of dM/dt in the limit oft_(P)=0 can be obtained graphically from a plot of M vs. t_(R) by doinga best fit of the curve and extrapolating to t_(R)=−t_(Q) . This wouldcorrespond to F_(RQ)=0 and C_(R)=0. Thus, Equation (38) would holdexactly and the Equation (37) would be written as

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t} = {V_{W}C_{D}\gamma_{1}}} & (39)\end{matrix}$As a result, the permeability would be given as

$\begin{matrix}{P = \frac{V_{W}\gamma_{1}}{A}} & (40)\end{matrix}$From the above, it is possible to obtain the diffusion coefficient D ofa drug in the dialysate medium. Equations (8), (9) and (17) lead to

$\begin{matrix}{\lambda = \frac{\beta_{1}{J_{1}\left( \beta_{1} \right)}}{J_{0}\left( \beta_{1} \right)}} & (41) \\{\gamma_{1} = \frac{\beta_{1}^{2}D}{a^{2}}} & (42) \\{\frac{P}{\gamma_{1}a} = \frac{J_{1}\left( \beta_{1} \right)}{\beta_{1}{J_{0}\left( \beta_{1} \right)}}} & (43)\end{matrix}$Knowing P, a and γ₁ allows to be calculated from Equation (43), whichthen allows D to be calculated from Equation (42) and λ from Equation(9) or Equation (41).

As mentioned above, the volume of the probe window must be accuratelyknown to obtain accurate values of the F_(RP). Since opticalmeasurements and using manufacturers' nominal specifications are notaccurate enough, the methods presented here were developed to moreaccurately determine V_(W).

If a long resting time is used in a PMD experiment, then theconcentrations of the dialysate resting in the probe window and in themedium outside the probe will equilibrate, soF_(RP)→1 long t_(R)  (44)and Equation (24) becomes

$\begin{matrix}{F_{R} = {F_{RQ} + {{V_{W}\left( {1 - F_{RQ}} \right)}\frac{1}{V_{S}}}}} & (45)\end{matrix}$A plot of F_(R) vs. 1/V_(S) will give an intercept of F_(RQ) and a slopeof V_(W)(1−F_(RQ)), which will allow the calculation of V_(W). (F_(RQ)can also be measured independently from CFMD data.) A variation of thismethod that will not require long resting times is to obtain a matrix ofF_(R) vs. 1/V_(S) for a range of resting times, and perform a nonlinearregression on the matrix. However, the preferred method of usingEquation (45) is preferred because it avoids the potential numericalproblems associated with nonlinear regressions.

Another variation that would also avoid the need for long resting timesis as follows. If all samples are taken in the same way, V_(S) andF_(RQ) are constant, and V_(W) does not change. Thus, rewriting Equation(24) as

$F_{R} = {{\frac{V_{W}}{V_{S}}F_{RP}} + {constant}}$and taking the derivative gives

$\begin{matrix}{\frac{\mathbb{d}F_{R}}{\mathbb{d}t_{R}} = {\frac{V_{W}}{V_{S}}\frac{\mathbb{d}F_{RP}}{\mathbb{d}t_{R}}}} & (46)\end{matrix}$Combining this with Equation (28) and approximating δ₁=1, which is validfor microdialysis probes, gives

$\frac{\mathbb{d}F_{R}}{\mathbb{d}t_{R}} = {\frac{V_{W}\gamma_{1}}{V_{S}}{\exp\left( {{- \gamma_{1}}t_{Q}} \right)}{\exp\left( {{- \gamma_{1}}t_{R}} \right)}}$This derivative is always positive, and taking the natural log gives

$\begin{matrix}{{\ln\left( \frac{\mathbb{d}F_{R}}{\mathbb{d}t_{R}} \right)} = {{{- \gamma_{1}}t_{R}} + k^{\prime}}} & (47) \\{k^{\prime} = {{{- \gamma_{1}}t_{Q}} + {\ln\left( \frac{V_{W}\gamma_{1}}{V_{S}} \right)}}} & (48)\end{matrix}$where k′ is a constant. From a plot of the natural log of the derivativevs. the resting time in Equation (47), γ₁ is obtained from the slope andV_(W) can be obtained from the intercept. It should be noted, however,that this method gives good results for γ₁ but is not as good forobtaining V_(W). In practice, V_(W) is best obtained from Equation (24)by plotting F_(R) vs. 1/V_(S) (described in Example 1 below). This canbe then be used when obtaining γ₁ by taking the slope of Equation (47),subject to the constraint that the intercept k′ be consistent with thevalue of V_(W) obtained from the plot of Equation (24). This is easilydone in Microsoft Excel® (using the Solver function) and other programs.

It should be noted that Equation (47) provides an alternative method fordetermining the parameter γ₁ from the slope of the log (dF_(R)/dt_(R))vs. t_(R) or tp (since t_(P)=t_(R)+t_(Q) leads to dt_(P)=dt_(R) whent_(Q) is constant). In practice, this method works well. It is typicallydone by fitting the F_(R) vs. t_(R) data to an empirical function(usually bi-exponential plus a constant), taking the derivativeanalytically, and then plotting the log of the derivative vs. t_(R).

From the above, it is possible to use PMD to characterize the volume ofthe probe window, its permeability coefficient for any drug, and theparameter λ. Some example methods are described below.

The donor Medium is Inside the Probe (Dialysate)

When the donor is inside the probe, C_(D) corresponds to theconcentration in the dialysate near the probe wall (r=a) and C_(R) isthe concentration in the medium outside the probe. If the receiverconcentration is always close to zero (sink conditions), and if theconcentration of drug (or other material to be separated) in thedialysate before entering the probe is denoted by C₀, then the boundaryand initial conditions that must be used in solving Equation (4) aregiven as

$\begin{matrix}{\begin{matrix}{{IC}\text{:}} & {{C\left( {r,0} \right)} = C_{0}} & {t = 0} \\{{BC}\text{:}} & {{{- D}\frac{\partial C}{\partial r}} = {PC}} & {r = a} \\\; & {{C\left( {0,t} \right)} = {finite}} & {r = 0}\end{matrix}\quad} & (49)\end{matrix}$Using the separation of variables method, Equations (4) and (49) can besolved to give the concentration in a volume element of dialysate at agiven radius and time as

$\begin{matrix}{{C\left( {r,t} \right)} = {2C_{0}{\sum{\frac{\lambda\;{J_{0}\left( {B_{n}{r/a}} \right)}}{\left( {\beta_{n}^{2} + \lambda^{2}} \right){J_{0}\left( \beta_{n} \right)}}{\exp\left( {- \frac{\beta_{n}^{2}{Dt}}{a^{2}}} \right)}}}}} & (50)\end{matrix}$where the symbols have the same definitions as those given previously.The same considerations outlined above hold, so the approximation ofEquation (34) leads to

${C\left( {r,t} \right)} = {\frac{2C_{0}\lambda\;{J_{0}\left( {B_{1}{r/a}} \right)}}{\left( {\beta_{1}^{2} + \lambda^{2}} \right){J_{0}\left( \beta_{1} \right)}}{\exp\left( {{- \gamma_{1}}t} \right)}}$Following steps analogous to those presented above (for the donoroutside the probe setup) leads to a set of equations analogous to theones given for that case. In this case, the quantity of interest is themass of drug that is left in the dialysis sample, which is given by M=M_(P)+M_(Q), whereM _(P) =V _(W) C ₀exp(−γt _(P))M _(Q)=(V _(S) −V _(W))C ₀ exp(−γ₁ t _(Q))It is useful to define the fraction of drug remaining in the dialysatesample by the term R_(F), given by Equation (2), which is analogous tothe F_(R) term discussed in the previous section. It is also possible todefine the fraction remaining from the pulsed and continuous portions,R_(FP) and R_(FQ), respectively, as

$\begin{matrix}{R_{FP} = {\frac{M_{P}}{V_{W}C_{0}} = {\exp\left( {{- \gamma_{1}}t_{P}} \right)}}} & (51) \\{{R_{FQ} = {\frac{M_{P}}{\left( {V_{S} - V_{W}} \right)C_{0}} = {\exp\left( {{- \gamma_{1}}t_{Q}} \right)}}}{{which}\mspace{14mu}{leads}\mspace{14mu}{to}}} & (52) \\{R_{F} = {{\frac{V_{W}}{V_{S}}R_{FP}} + {\frac{V_{S} - V_{W}}{V_{S}}R_{FQ}}}} & (53)\end{matrix}$

It is possible to find the R_(FQ) in a manner analogous to given inEquations (26) and (27) by fitting the R_(F) vs. t_(R) data to anempirical function using nonlinear regression, and then taking the valueof the function for t_(R)=0, using the equations below:R _(F)=1−a ₁ exp(−b ₁ t _(R))−a ₂ exp(−b ₂ t _(R))  (54)R _(FQ)=1−a₁ −a ₂  (55)Following a mathematical analysis that is similar to that leading toEquations (30) and (47) leads to the following equations that can beused to obtain γ₁:

$\begin{matrix}{{\ln\mspace{14mu} R_{FP}} = {{- \gamma_{1}}t_{P}}} & (56) \\{{\ln{\frac{\mathbb{d}R_{F}}{\mathbb{d}t_{r}}}} = {{{- \gamma_{1}}t_{R}} + {\ln\left( \frac{\gamma_{1}V_{W}}{V_{S}} \right)} - {\gamma_{1}t_{Q}}}} & (57)\end{matrix}$It should be noted that R_(F) decreases with increasing t_(R), so thelog is taken of the absolute value of the derivative. (This is reflectedin the right hand side of Equation (57) as well.)Equations for the permeability of the probe wall, and thus λ, can befound as follows. Following logic analogous to that presented for thedonor in the medium outside the probe, if the receiver concentration isconstant and can be approximated as zero, then the initial rate of lossfrom the dialysate (at t_(P) approaching zero, which is equivalent tot_(R)=−t_(Q)), is

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t_{R}} = {- {APC}_{0}}} & (58)\end{matrix}$where the (−)sign signifies that the drug is lost from the dialysate.From Equations (51) and (53),

$\frac{\mathbb{d}M}{\mathbb{d}t_{R}} = {C_{0}\frac{V_{W}}{V_{S}}\frac{\mathbb{d}R_{FP}}{\mathbb{d}t_{R}}}$which leads to

$\frac{\mathbb{d}M}{\mathbb{d}t_{R}} = {{- V_{W}}C_{0}\gamma_{1}{\exp\left( {{- \gamma_{1}}t_{Q}} \right)}{\exp\left( {{- \gamma_{1}}t_{R}} \right)}}$When t_(R)=−t_(Q), this gives

$\begin{matrix}{\frac{\mathbb{d}M}{\mathbb{d}t_{R}} = {{- V_{W}}C_{0}\gamma_{1}}} & (59)\end{matrix}$Combining Equations (58) and (59) gives the permeability as

$\begin{matrix}{P = \frac{V_{W}\gamma_{1}}{A}} & (60)\end{matrix}$which is the same as Equation (40).Pulsatile Microdialysis: Mathematical Model when the Donor Medium isOutside the Probe and the Donor Concentration is Rapidly Changing

It is sometimes of interest to measure concentrations in the mediumoutside the probe for cases in which the concentration of free drugchanges rapidly. This can occur with protein binding and otherprocesses. One problem associated with such rapidly changing systems isobtaining samples often enough to characterize the donor concentrationover time. However, if the concentration in the medium outside the probe(which will be considered as the donor here) changes significantlyduring the collection of a PMD sample, then it becomes necessary todetermine both a donor concentration and the time at which the donor hadthat concentration. The concentration in a PMD sample is a reflection ofthe mass taken up during the entire sampling interval and does not givespecific details about the rate of mass accumulation at any specifictime in the interval. Thus, an extension of the analysis in the previoussection (for constant external medium concentration) is needed to find aspecific donor concentration that occurs at a specific time. This is nota problem when the donor concentration changes very slowly or is static,but must be addressed for the fast systems mentioned above.

In practice, each PMD sample is a combination of three portions orsub-samples—one that is initially pumped through the probe windowwithout resting, followed by one that is pumped into the probe windowand then allowed to remain at rest for a given time (known as theresting time t_(R)), and a final portion that is used to flush the probewindow without resting. Sub-samples are categorized according to theexposure time of the dialysate (i.e., time spent in the probe window).Within each sub-sample, all small volume elements of fluid are exposedto the donor for the same time At, but At will not be the same for anytwo consecutive sub-samples. This is important because the exposure timewill affect how much drug is accumulated in the dialysate. In addition,not all parts of the dialysate enter the probe window at the same time,which must be considered when the donor concentration changes with time.

Below, the mass taken up by a PMD sample will be calculated in terms ofthe free donor concentration (dissolved and not bound or adsorbed) andrelevant PMD parameters. This will be done for an arbitrary PMDsub-sample by calculating the rate of drug accumulation in aninfinitesimal volume element of dialysate, integrating over the entireexposure interval to find the mass accumulated by the element while inthe probe window, and then summing the masses for all elements to findthe total mass in the sub-sample. Subsequently, this analysis will beapplied to each PMD sub-sample and the mass accumulated in a completePMD sample will be found. Two cases will then be considered—when thedonor concentration (free drug) is constant (static donor), and when thedonor concentration changes with time (dynamic donor).

The Mass Accumulated in a PMD Sub-Sample

For a constant donor concentration C_(D), the mass of drug in a PMDsub-sample is given by Equation (15) or (16). Using the Equation (34)and denoting the exposure time in the sub-sample (either t_(Z) or t_(P))by Δt, the concentration of drug in the PMD sub-sample is given byC=C _(D)(1−exp[−γ₁ Δt])  (61)Here, as before, γ characterizes how quickly the concentration in thedialysate approaches equilibration with the donor and can be determinedexperimentally (discussed below).

If the concentration in the donor is not constant, then Equation (61) nolonger holds because different parts of the sample enter the probewindow at different times, and are thus exposed to different donorconcentrations. Instead, the instantaneous rate of accumulation must beconsidered. Here, we will consider an infinitesimal volume element ofdialysate that enters the probe window between times t′ and t′+dt′, hasa infinitesimal volume dV=Qdt′ , and has been within the probe windowfor a time t_(E)(0≦t_(E)≦Δt). While the given volume element is in theprobe window, the donor concentration is a function of the time when theelement is in the probe, given as t=t′+t_(E). Thus, the rate of changeof concentration in that volume element is

$\frac{\mathbb{d}C}{\mathbb{d}t} = {\gamma_{1}{C_{D}\left( {t^{\prime},t_{E}} \right)}{\exp\left\lbrack {{- \gamma_{1}}t_{E}} \right\rbrack}}$and the mass of drug in the volume element (denoted by m=C dV)accumulates at a rate

$\begin{matrix}{\frac{\mathbb{d}m}{\mathbb{d}t} = {{\frac{\mathbb{d}C}{\mathbb{d}t}{dV}} = {\gamma_{1}{C_{D}\left( {t^{\prime},t_{E}} \right)}{\exp\left( {{- \gamma_{1}}t_{E}} \right)}{Qdt}^{\prime}}}} & (62)\end{matrix}$The mass accumulated in the volume element while it is in the probewindow is found by integrating Equation (62) over the exposure intervalt_(E)=0 to Δt, or

$m = {{dt}^{\prime}{\int_{0}^{\Delta\; t}{\frac{\mathbb{d}m}{\mathbb{d}t}\ {\mathbb{d}t_{E}}}}}$The total mass in the sample is found by adding the mass accumulated inall the volume elements, which is done by integrating over all entrytimes to give

$\begin{matrix}{M = {{\int_{t_{0}}^{t_{f}}{m\ {\mathbb{d}t^{\prime}}}} = {\int_{t_{0}}^{t_{f}}\ {{\mathbb{d}t^{\prime}}{\int_{0}^{\Delta\; t}{\frac{\mathbb{d}m}{\mathbb{d}t}\ {\mathbb{d}t_{E}}}}}}}} & (63)\end{matrix}$where t₀ and t_(f) denote the entry time into the probe window for thefirst and last volume elements of the sub-sample, respectively.Application to a Complete PMD Sample

As mentioned above, a complete PMD sample is a combination threesub-samples, each with its own exposure time Δt. In general, theexposure time Δt=t_(Q)+t_(R), where t_(R) is the chosen resting time andt_(Q) is the transit time given by Equation (13). In addition, not allparts of the PMD sample enter the probe window at the same time, sodifferent parts of each sample and sub-sample are in the probe window atdifferent times and are therefore exposed to different donorconcentrations. In what follows, the start time for the PMD sample willbe denoted as t⁰, which will be taken as the time when the flushing ofthe previous sample (its last sub-sample) stops. In addition, forsub-sample n (n=1,2,3) of the PMD sample, the times at which the firstand last volume elements enter the probe window are denoted as t_(0,n)and t_(f,n), respectively. Finally, it is assumed that all flushing isdone at the same flush rate Q (volume/time). Using this convention, thedetails of each sub-sample are as follows:

-   -   Sub-sample 1, which is initially pumped through the probe window        without resting, corresponds to the residual dialysate that is        left in the outlet tubing after flushing out the previous        sample. For this portion, the mass collected is M₁ and the        volume V₁ is the volume of the outlet tubing. The times at which        the first and last volume elements of the sub-sample enter the        probe window, t_(0,1) and t_(f,1), and the exposure time for        each volume element Δt₁ are        t _(0,1) =t ⁰−(t _(Q) +V ₁ /Q) t _(f,1) =t ⁰ −V ₁ /Q Δt=t        _(Q)  (64)    -   Sub-sample 2, which follows the first one, is pumped into the        probe window, allowed to occupy the window at rest for a        specified resting time tR, and then flushed out by sub-sample 3.        For this portion, the mass collected is M2 and the volume V2=VW        (the probe window volume). The time t_(f,2) corresponds to the        time when the flushing of the previous sample stops. The times        at which the first and last volume elements of the sub-sample        enter the probe window, and the exposure time for each volume        element are        t _(0,2) =t ⁰ −t _(Q) t_(f,2) =t ^(0 Δt) ₂ =t _(R) +t _(Q)  (65)    -   Sub-sample 3 is the final portion, and is used to flush the        probe without resting to facilitate a more complete collection        of the pulsed portion. For this portion, the mass collected is        M₃ and the volume is V₃=V_(S)−V₁−V₂. The times at which the        first and last volume elements of the sub-sample enter the probe        window, and the exposure time for each volume element are        t _(0,3) =t ⁰ +t _(R) t _(f,3) =t ⁰ +t _(S)−(t _(Q) +V ₁ /Q)        Δt₃=t_(Q)  (66)

The total mass of drug in the PMD sample is M=M₁+M₂+M₃, and the totalsample volume is V_(S)=V₁+V₂+V₃. (It should be noted that the total timeto collect the sample, denoted by t_(S), is not the same as the sum ofthe exposure times.)

Mass Accumulated in a PMD Sample When the Donor Concentration isConstant (Static)

When the donor concentration is constant, Equation (62) becomes

$\frac{\mathbb{d}m}{\mathbb{d}t} = {{\frac{\mathbb{d}C}{\mathbb{d}t}{dV}} = {\gamma_{1}C_{D}{\exp\left( {{- \gamma_{1}}t_{E}} \right)}{Qdt}^{\prime}}}$Performing the integrations in Equation (63) gives the mass in thesub-sample n asM _(n) =C _(D) V _(n)[1−exp(−γ₁ Δt _(n))]where the sub-sample volume V_(n) is given asV _(n) =Q[(t ⁰ +t _(f,n))−(t ⁰ +t _(0,n))]=Q(t _(f,n) −t _(0,n))Summing the mass over the three sub-samples gives the mass in the totalPMD sample as

$\begin{matrix}{M = {\sum\limits_{n = 1}^{3}{V_{n}{C_{D}\left\lbrack {1 - {\exp\left( {{- \gamma_{1}}\Delta\; t_{n}} \right)}} \right\rbrack}}}} & (67)\end{matrix}$Experimentally, the mass in the in the PMD sample is found fromM=V_(S)C_(S)  (68)where C_(S) is the concentration of drug in the PMD sample. Thus, thefraction recovered F_(R) for the PMD sample is given by

$\begin{matrix}{F_{R} = {\frac{C_{S}}{C_{D}} = {\frac{1}{V_{S}}{\sum\limits_{n = 1}^{3}{V_{n}\left\lbrack {1 - {\exp\left( {{- \gamma_{1}}\Delta\; t_{n}} \right)}} \right\rbrack}}}}} & (69)\end{matrix}$

Knowing the sub-sample volumes and the F_(R) (which is found from theslope of a C_(S) vs. C_(D) plot), Equation (69) can be used to obtainγ₁.

Mass Accumulated in a PMD Sample When the Donor Concentration isChanging (Dynamic)

When the donor concentration is changing, C_(D) must now be representedas a function of time. In practice, two factors influence how C_(D) isrepresented. First, it should be possible to integrate and differentiatethe function (analytically or numerically. Second, the function shouldbe of a form that is suited to represent the anticipated experimentaldata (usually based on a physical model).

In this work, two systems were studied—a simulated first order declinein donor concentration, and the protein binding. For both cases, the(free) drug concentration in the donor over the entire experiment wasmodeled as a function of time using a bi-exponential function plus aconstant, of the formC _(D) =a ₁exp(−b ₁ t)+a ₂exp(−b₂ t)  (70)(This choice of function was motivated in part by the protein bindingkinetics model presented below. It is also appropriate for the firstorder experiments, with a₂ and a₃→0.) Here, one set of fittingparameters (a_(j), b_(j)) is used to characterize the time behavior ofC_(D) over the entire time of the experiment. It is important to notethat these parameters reflect processes affecting the donorconcentration, and are independent of the probe as well. In addition,the fitting parameters do not have to have physical significance, butare only required to fit the data numerically. Equation (70) can bewritten more compactly as

$\begin{matrix}{C_{D} = {\sum\limits_{j = 1}^{3}{a_{j}{\exp\left( {{- b_{j}}t} \right)}}}} & (71)\end{matrix}$(The constant term is achieved by letting b₃→0.) With this choice,Equation (62) gives

$\frac{\mathbb{d}m}{\mathbb{d}t} = {\gamma_{1}Q\;{\exp\left\lbrack {{- \gamma}\; t_{E}} \right\rbrack}{\sum\limits_{j = 1}^{3}{a_{j}{\exp\left\lbrack {- {b_{j}\left( {t^{\prime} + t_{E}} \right)}} \right\rbrack}{\mathbb{d}t^{\prime}}}}}$If all PMD samples are collected using the same procedure, then for agiven n, Δt_(n) is the same for all i. (In other words, Δt₁ is the samefor all PMD samples, etc.), and Equation (63) gives the mass insub-sample n of the i^(th) PMD sample as

$\begin{matrix}{M_{n} = {\sum\limits_{j = 1}^{3}{\left( \frac{Q\;\gamma_{1}a_{j}}{b_{j}\left( {b_{j} + \gamma_{1}} \right)} \right) \times \left\{ {1 - {\exp\left\lbrack {{- \left( {b_{j} + \gamma_{1}} \right)}\Delta\; t_{n}} \right\rbrack}} \right\} \times {\quad\left\{ {{\exp\left\lbrack {- {bt}_{0,n}} \right\rbrack} - {\exp\left\lbrack {- {bt}_{0,f}} \right\rbrack}} \right\}}}}} & (72)\end{matrix}$The total mass in the PMD sample is given byM=M ₁ +M ₂ +M ₃  (73)and the overall concentration in the PMD sample is given by

$\begin{matrix}{C_{S} = \frac{M}{V_{S}}} & (74)\end{matrix}$

Once the PMD sample concentration C_(S) is obtained, it is possible toplot the donor concentration at any time within the sampling interval.However, it is most convenient to calculate C_(D) at the start time ofthe PMD sample t⁰. For comparison with the static calibrations, it ispossible to define another fractional recovery, denoted as F_(R) ⁰ asthe ratio of the C_(S) to the donor concentration at the start of theinterval, or

$\begin{matrix}{F_{R}^{0} = \frac{C_{S}}{C_{D}\left( t^{0} \right)}} & (75)\end{matrix}$

As mentioned above, the time dependence of C_(D) over the time of theexperiment is characterized by the set of fitting parameters (a_(j),b_(j)). The flush rate Q is selected before the experiment, thesub-sample volumes are known (thus allowing the time parameters to becalculated using Equations (64)-(66)), and γ₁ can be determinedexperimentally from static data using Equation (69). Thus, if enough PMDsamples are collected, the values of a_(j) b_(j), and can be found by anonlinear regression analysis, in which Equations (72)-(74) are fit tothe PMD data.

It should be noted that, when the donor concentration is constant,Equations (72)-(74) give the same result as Equation (67), and F_(R)⁰=F_(R). Mathematically, this is done by requiring that Σa_(i)=C_(D),and setting the b_(i)=0 (which is done by first expanding theexponentials and then taking the limit as b→0.)

Numerical Procedure

The implementation of the above equations is described below andillustrated in the Examples. Fitting functions for the data should becarefully chosen, with the following guidelines in mind. First, thefitting function should be smooth and differentiable, and capture theessential features of the physical data (i.e., maxima/minima and theasymptotic behavior). In addition, if a mathematical model is being usedto analyze the experimental data, the fitting function must have atleast as many degrees of freedom (i.e., fitting parameters a_(i) andb_(j)) as the basic physical equations. Further, good fitting practicesshould be followed (more data points than fitting parameters, sufficientnumber of points to characterize any curvatures, etc.). The procedure isas follows:

-   -   For the probe or combination of probes being used, the probe or        set of probes should be calibrated for the static F_(R)        (constant concentration in the medium outside the probe or        probes) for a given resting time, and using a number of        different sample volumes.    -   From the static F_(R), determine the value of γ₁ for the probe        or combination of probes using Equation (69). (Alternatively, γ₁        can be obtained from F_(R) vs. t_(R) data, as described below.)        However, when the donor concentration is changing, it is        preferable to use this method because it reflects the behavior        of a particular probe or set of probes in this context.)    -   From the experiments in which the donor concentration is        changing, fit the PMD data in terms with the empirical function        given by Equation (70) or (71) using Equations (72)-(74).    -   Take the plot times as the beginning of each PMD sample (i.e.,        when the flushing from the previous sample has stopped), and        calculate the corresponding concentration in the donor using the        F_(R) ⁰ given by Equation (75).

EXAMPLE 1 Determination of F_(RQ) when the Donor Medium is Outside theProbe and the Donor Concentration is Constant.

It is possible to measure the F_(RQ) directly using CFMD data.Alternatively, it is possible to obtain this parameter using PMD data.In this application, the donor is outside the probe, and itsconcentration is taken as constant (so the F_(RQ) is well defined).

An example of a method that uses PMD to determine F_(RQ) is as follows:

-   a) Immerse a probe in a solution (medium outside the probe)    containing a known concentration of drug. The volume of the external    solution should be large enough (at least ˜25 mL) so drug transfer    to the dialysate will not change the external medium concentration.-   b) Pump fresh dialysate (i.e., containing no drug) into the probe    window. The dialysate should be the same liquid as the external    medium. It is preferable that the flow rate Q be relatively high (at    least ˜100 μL/min), so that F_(RQ) is much smaller than 1.-   c) Allow the dialysate to occupy the probe at rest for a known time    t_(R).-   d) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. V_(S) must be sufficient to collect all of the    dialysate that was at rest in the window.-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample, and    calculate the F_(R) from Equation (1).-   f) Repeat steps b) through e) using the same sample volume and flow    rate, but for at least one (preferably at least four) different    resting times.-   g) Fit a plot of the F_(R) vs. t_(R) to the empirical function given    by Equation (26).-   h) Set t_(R)=0 and calculate F_(RQ) from Equation (27).

EXAMPLE 2 Determination of R_(FQ) when the Donor Medium is Inside theProbe and Concentration Outside the Probe is Zero

Here, R_(FQ) will be obtained using PMD data. In this application, thedialysate serves as the donor and the medium surrounding the probe isthe receiver with a constant concentration. (In other words, the masslost by the dialysate does not significantly change the receiverconcentration.)

An example of a method that uses PMD to determine R_(FQ) is as follows:

-   a) Immerse a probe in a solution (medium outside the probe)    containing a known concentration of drug. The volume of the external    solution should be large enough (at least ˜25 mL) so drug transfer    to the dialysate will not change the external medium concentration.-   b) Pump dialysate containing a known concentration of drug C₀ into    the probe window. The dialysate should be the same liquid as the    external medium. It is preferable that the flow rate Q be relatively    high (at least ˜100 μ/min), so that R_(FQ) is much smaller than 1.-   c) Allow the dialysate to occupy the probe at rest for a known time    t_(R).-   d) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. V_(S) must be sufficient to collect all of the    dialysate that was at rest in the window.-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample, and    calculate the R_(F) from Equation (2).-   f) Repeat steps b) through e) using the same sample volume and flow    rate, but for at least one (preferably at least four) different    resting times.-   g) Fit a plot of the R_(F) vs. t_(R) to the empirical function given    by Equation (54).-   h) Set t_(R)=0 and calculate R_(FQ) from Equation (55).

EXAMPLE 3 Determination of the Probe Window Volume

Measuring the window volume in this way is preferred since done in thecontext of the way the probe would actually be used, and also inherentlytakes into account any irregularities in the probe geometry which wouldgive somewhat erroneous results using other methods, such as opticalmeasurements.

An example of a method that uses PMD to determine V_(W) is as follows:

-   a) Immerse a probe in a solution (medium outside the probe)    containing a known concentration of drug. The volume of the external    solution should be large enough (at least ˜25 mL) so drug transfer    to the dialysate will not change the external medium concentration.-   b) Pump fresh dialysate (i.e., containing no drug) into the probe    window. The dialysate should be the same liquid as the external    medium. It is preferable that the flow rate Q be relatively high (at    least ˜100 μL/min), so that F_(RQ) is much smaller than 1.-   c) Allow the dialysate to occupy the probe at rest for a known    resting time t_(R). It is preferred that the resting time be long    enough (typically ˜310 minutes) so the F_(RP)≈1.-   d) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. V_(S) must be sufficient to collect all of the    dialysate that was at rest in the window.-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample, and    calculate the F_(R) from Equation (1).-   f) Repeat steps b) through e) using the same resting time and flow    rate, but for at least one (preferably at least three) different    sample volumes.-   g) Plot F_(R) vs. 1/V_(S), which will give a straight line (e.g.,    FIG. 7), and determine the slope and intercept.-   h) From Equation (24), the intercept will equal the F_(RQ) and the    slope will equal V_(W) (1−F_(RQ)) for long enough resting time.    Thus,

$V_{W} = {\frac{slope}{\left( {1 - F_{RQ}} \right)} = {\frac{slope}{\left( {1 - {intercept}} \right)}.}}$

EXAMPLE 4 Determination of γ₁.

Measuring the value of γ₁ in this way is one of the preferred waysbecause it does not require a knowledge of V_(W). However, knowledge ofV_(W) is still desirable because it can be used to improve the accuracyof the method.

An example of a method that uses PMD to determine γ₁ is as follows:

-   a) Immerse a probe in a solution (medium outside the probe)    containing a known concentration of drug. The volume of the external    solution should be large enough (at least ˜25 mL) so drug transfer    to the dialysate will not change the external medium concentration.-   b) Pump fresh dialysate (i.e., containing no drug) into the probe    window. The dialysate should be the same liquid as the external    medium. It is preferable that the flow rate Q be relatively high (at    least ˜100 μL/min), so that F_(RQ) is much smaller than 1.-   c) Allow the dialysate to occupy the probe at rest for a known time    t_(R).-   d) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. V_(S) must be sufficient to collect all of the    dialysate that was at rest in the window.-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample, and    calculate the F_(R) from Equation (1).-   f) Repeat steps b) through e) using the same sample volume and flow    rate, but for at least one (preferably at least four) different    resting times.-   g) Determine the F_(RQ) from CFMD data or the method of Example 1.-   h) Using the values of the sample volume and the window volume (such    as determined from Example 1), calculate the F_(RP) for each resting    time from Equation (24).-   i) Plot ln (1−F_(RP)) vs. t_(R) or t_(P). This plot will give a    straight line (e.g., FIG. 7) with a slope of γ₁.

EXAMPLE 5 Determination of γ₁.

Measuring the value of γ₁ in this way is one of the preferred waysbecause it does not require a knowledge of F_(RQ). It also does notrequire knowledge of V_(W), but knowing of V_(W) is desirable because itcan be used to improve the accuracy of the method.

An example of a method that uses PMD to determine γ₁ is as follows:

-   a) Immerse a probe in a solution (medium outside the probe)    containing a known concentration of drug. The volume of the external    solution should be large enough (at least ˜25 mL) so drug transfer    to the dialysate will not change the external medium concentration.-   b) Pump fresh dialysate (i.e., containing no drug) into the probe    window. The dialysate should be the same liquid as the external    medium. It is preferable that the flow rate Q be relatively high (at    least ˜100 μL/min), so that F_(RQ) is much smaller than 1.-   c) Allow the dialysate to occupy the probe at rest for a known time    t_(R).-   d) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. V_(S) must be sufficient to collect all of the    dialysate that was at rest in the window.-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample, and    calculate the F_(R) from Equation (1).-   f) Repeat steps b) through e) using the same sample volume and flow    rate, but for at least one (preferably at least four) different    resting times.-   g) Fit a plot of the F_(R) vs. t_(R) to the empirical function given    by Equation (26).-   h) Take the derivative of the fitted equation to find dF_(R)/dt_(R)    as a function of t_(R).-   i) Plot ln (dF_(R)/dt_(R)) vs. t_(R), as prescribed by Equation    (47). This plot will give a straight line (e.g., FIG. 7xxxx) with a    slope of γ₁. The accuracy can be improved if the plot is made    subject to the constraint that the intercept, given by Equation    (48), is consistent with the value of V_(W) found independently    using the method of Example 1, and value of γ₁ found from the slope.    (This would be done by an iterative procedure.)

EXAMPLE 6 Determination of The Probe Window Wall PermeabilityCoefficient: Drug Outside the Probe

The permeability P of the probe window wall is defined at steady stateby Equation (5), and measuring the rate at which a drug crosses themembrane will allow P to be determined. A simple method using PMD isdescribed below, using a setup in which a drug that is initially in theexternal medium crosses the probe wall membrane and transfers into thedialysate. (It is also possible to determine the permeability using asetup in which the drug is initially in the dialysate, and measuring therate at which it transfers into the external medium.)

-   a) Make a solution containing a known concentration of drug    (typically in water or a buffer), and use it as the external medium.    The volume of the external medium should be large enough (typically    at least ˜25 mL) so that the drug transferred to the dialysate will    not significantly change the external medium concentration C_(D).-   b) Use the same medium that initially contains no drug as the    dialysate. Immerse the probe into the external medium.-   c) At a high flow rate Q (at least 100 μL/min), pump the dialysate    into the probe.-   d) Allow the dialysate to occupy the probe for a known resting time    t_(R).-   e) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. It is preferred that V_(S) is sufficient to    collect all of the dialysate that was at rest in the window.-   f) Perform an appropriate assay (HPLC, etc.) to determine the amount    of drug in the dialysate sample.-   g) Repeat steps c) through f) using the same flow rate and sample    volume, but for at least one (preferably at least three) different    resting times. It is preferred that at least one or two of the    resting times be short, typically in the range of ˜2-5 times the    transit time t_(Q), defined in Equation (13).-   h) Plot the amount of drug in the dialysate sample vs. the resting    time. The slope is dM/dt.-   i) Determine the slope using points corresponding to small values of    t_(R). (This corresponds to accumulating only small amounts of drug    in the dialysate and allows the approximation to be made that the    concentration in the dialysate is much smaller than the    concentration C_(D) in the external medium, so Equation (38) can be    used.)-   j) Determine the area of the probe window (from its length and    radius), and calculate the permeability using

$P = {\frac{1}{{AC}_{D}}{\frac{\mathbb{d}M}{\mathbb{d}t}.}}$

EXAMPLE 7 Determination of the Probe Window Wall PermeabilityCoefficient: Drug Inside the Probe

The permeability P of the probe window wall is defined at steady stateby Equation (5), and measuring the rate at which a drug crosses themembrane will allow P to be determined. A simple method using PMD isdescribed below, using a setup in which a drug that is initially in thedialysate crosses the probe wall membrane and transfers into an externalmedium. (It is also possible to determine the permeability using a setupin which the drug is initially in the external medium, and measuring therate at which it transfers into the dialysate.)

-   a) Make a solution containing a known concentration of drug    (typically in water), and use it as the dialysate. The volume of the    external medium should be large enough (typically at least 25 mL) so    that the drug transferred from the dialysate will not significantly    change the external medium concentration.-   b) Immerse the probe in a volume of the same liquid used in the    dialysate that initially contains no drug. The volume of liquid    outside of the probe should be large enough (typically at least ˜25    mL) so that the drug concentration in the external medium is always    negligible compared to the dialysate drug concentration. This allows    the approximation C_(D)−C_(R)=C_(D) in Equation (5), where C_(D)    represents the dialysate drug concentration.-   c) At a high flow rate Q (at least ˜100 μL/min), pump the dialysate    into the probe.-   d) Allow the dialysate to occupy the probe for a known resting time    t_(R).-   e) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. It is preferred that V_(S) is sufficient to    collect all of the dialysate that was at rest in the window.-   f) Perform an appropriate assay (HPLC, etc.) to determine the amount    of drug left in the dialysate sample. Knowing the initial    concentration and amount in the sample volume, calculate the amount    of drug that was lost from the dialysate into the external medium.-   g) Repeat steps c) through f) using the same flow rate and sample    volume, but for at least one (preferably at least three) different    resting times. It is preferred that at least one or two of the    resting times be short, typically in the range of 2-5 times the    transit time t_(Q), defined in Equation (13).-   h) Plot the amount lost from the sample vs. the resting time. The    slope is dM/dt.-   i) Determine the slope using points corresponding to small values of    t_(R). (This corresponds to losing only small amounts of drug from    the dialysate and allows the approximation be made that C_(D) is    constant, so Equation (38) can be used.)-   j) Determine the area of the probe window (from its length and    radius), and calculate the permeability using

$P = {{- \frac{1}{{AC}_{D}}}{\frac{\mathbb{d}M}{\mathbb{d}t}.}}$

EXAMPLE 8 Determination of the Window Wall Permeability Coefficient:Drug Outside the Probe

An alternative method for determining P is based on transferring thedrug from the external medium into the dialysate. A simple example isdescribed below.

-   a) Immerse the probe in a solution (medium outside the probe)    containing a known concentration of the drug. The volume of the    external solution should be large enough (at least ˜25 mL) so drug    transfer to the dialysate will not change the external medium    concentration.-   b) At a high flow rate Q (at least ˜100 μL/min), pump the dialysate    through the probe continuously (i.e., CFMD) and collect a relatively    large sample volume (typically 20-50 μL).-   c) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample. Calculate    the F_(RQ) from Equation (1)-   d) At the same flow rate Q, pump the dialysate into the probe.-   e) Allow the dialysate to occupy the probe for a known resting time    t_(R).-   f) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. It is preferred that V_(S) be sufficient to    collect all of the dialysate that was at rest in the window.-   i) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample. Calculate    the F_(R) from Equation (1) and F_(R)p from Equation (24), using the    F_(RQ) determined in steps d) and e).-   g) Repeat steps f) through j) using the same flow rate and sample    volume, but for at least one (preferably at least three) different    resting times.-   h) Plot ln (1−F_(RP)) vs. t_(P), which will give a straight line    according to Equation (31). The slope of the line will equal −γ₁.-   i) Determine the window volume, as described in Example 3.-   j) Calculate the permeability P from Equation (40).

EXAMPLE 9 Determination of D and λ

The parameter λ characterizes the transfer across the probe window andhow that transfer affects the concentration of the agent in thedialysate. Preferably, λ is 0-10, more preferably 0-3, still morepreferably <0.3.

An example method to determine the parameter λ for a probe using PMD isas follows:

-   a) Make a solution containing a known concentration of a drug in a    liquid, and the external donor medium. The diffusion coefficient    does not need to be known.-   b) Follow the steps outlined in Example 1 to obtain the window    volume V_(W). From that value, and the length of the probe, obtain    the probe radius a.-   c) Follow steps b) through i) in the method to determine the    permeability coefficient P for the probe (Example 2 or 3).-   d) Once P has been determined, calculate β₁ from Equation (43)    (using the value of γ₁ obtained from Example 2 or 3 while getting P.-   e) From β₁, calculate D from Equation (42).-   f) From β₁, calculate λ from Equation (9) or Equation (41)

EXAMPLE 10 Determination of λ

The parameter λ characterizes the transfer across the probe window andhow that transfer affects the concentration of the agent in thedialysate. Preferably, λ is 0-10, more preferably 0-3, still morepreferably <0.3.

An example method to determine the parameter λ for a probe using PMD isas follows:

-   a) Make a solution containing a known concentration of reference    drug in a liquid, and use it as the dialysate. The diffusion    coefficient of the reference drug in the dialysate must be known    independently of this method (i.e., from the literature or measured    in some other experiment).-   b) Follow steps b) through i) in the method to determine the    permeability coefficient P for the probe (Example 2 or 3).-   c) Once P has been determined, calculate λ using Equation (9).

EXAMPLE 11 Determination of Diffusion Coefficient

If the probe has been characterized so that V_(W) and λ are known, it ispossible to determine the diffusion coefficient of any drug in thedialysate. A method of determining the diffusion coefficient of drugs inthe dialysate is as follows.

-   a) Determine the probe window volume V_(W) (for instance, using the    procedure described above). Any drug can be used.-   b) Using a reference drug, determine the value of λ (for instance,    using the procedures described above).-   c) Immerse the probe in a solution (medium outside the probe)    containing a known concentration of the drug for which D is to be    determined. The volume of the external solution should be large    enough (at least ˜25 mL) so drug transfer to the dialysate will not    change the external medium concentration.-   d) At a high flow rate Q (at least ˜100 μL/min), pump the dialysate    through the probe continuously (i.e., CFMD) and collect a relatively    large sample volume (typically ˜20-50 μL).-   e) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample. Calculate    the F_(RQ) from Equation (1)-   f) At a high flow rate Q (same one used for CFMD above), pump the    dialysate into the probe.-   g) Allow the dialysate to occupy the probe for a known resting time    t_(R).-   h) Flush and collect a known sample volume V_(S) of the dialysate at    the same flow rate Q. It is preferred that V_(S) is sufficient to    collect all of the dialysate that was at rest in the window.-   j) Perform an appropriate assay (HPLC, etc.) to determine the    concentration and amount of drug in the dialysate sample. Calculate    the F_(R) from Equation (1) and F_(RP) from Equation (24), using the    F_(RQ) determined in steps d) and e).-   i) Repeat steps f) through j) using the same flow rate and sample    volume, but for at least one (preferably at least three) different    resting times.-   j) Plot In (1−F_(RP)) vs. t_(P), which will give a straight line    according to Equation (31). The slope of the line will equal −γ₁.-   k) Using the value of λ for the probe, calculate β₁ from Equation    (33), then calculate the diffusion coefficient D using Equation    (31).

EXAMPLE 12 General Description of PMD for Uptake Into Dialysate

The PMD method was tested using methazolamide (Sigma Chemical, St.Louis, Mo.) as the test drug in a variety of in vitro experiments.

Microdialysis probes were constructed as follows. Hollow fibers made ofreconstituted cellulose (Spectrum Laboratory, Brunswick, N.J.), with amolecular cut-off of ˜18,000 Daltons (18 KD) were used as the dialysismembrane. The inner radius of the dialysis membrane tube was ˜100 μm andthe thickness of the membrane wall was ˜8 μm. A segment of polyimidetubing (MicroLumen, Tampa, Fla.) with an outer radius of ˜83 μm wasconnected to each end of the dialysis membrane and glued to it bycyanoacrylate glue, leaving a probe window of ˜10 cm length. (It will beunderstood by those skilled in the art that a straight probe, comprisinga straight, needle-type probe comprising a highly permeable tubularmembrane concentrically positioned within an impermeable tube (such asthose readily available from CMA Microdialysis AB, North Chelmsford,Mass.) could optionally also be used.) One segment (7 cm length) wasthen glued to a 15 cm Tygon® tube with a inner diameter of ˜100 μm,which was connected to a syringe pump and served as an inlet tubing. Theother segment (10 cm length) was used to collect the samples. Aschematic diagram of such a probe is shown in FIG. 1. A Harvard ModelPHD2000 programmable syringe pump (Harvard Apparatus, Holliston, MA) wasused to pump 0.005N NaOH dialysate through the probe. A jacketed beakerwith a magnetic stirrer was used as the in vitro donor compartment.Different concentrations of methazolamide (MTZ) solution in 0.005N NaOH(50 mL volume) were used as the donor medium. The dialysis membrane partof the probe was immersed in the donor medium and held stationary with aclamp. The MTZ solution was stirred continuously to keep a constantconcentration at the fluid-membrane boundary. All studies were performedat 37° C. The outer tubing (10 cm length) was used to collect thedialysate into 0.25 mL pre-chilled plastic micro-centrifuge tubes. Thesamples were analyzed immediately by high-pressure liquid chromatography(HPLC). (Preliminary studies found no evidence of non-specific bindingor adsorption of MTZ to the sampling tubes.)

MTZ was analyzed by HPLC with UV detection, according to the methoddescribed in J. Pharmocokin. Biopharm., 27(1): 45-66 (1999). The HPLCsystem consisted of a Shimadzu LC-10AD constant flow pump and SPD-10VAultraviolet detector (Shimadzu, Piscataway, N.J.), and a column. Outputfrom the detector was processed on a personal computer using TC4Turbochrom HPLC software (Perkin-Elmer, Shelton, Conn.). Separation wasaccomplished with a Pondapak 300 mm×3.9 mm C18, column (WatersCorporation, Milford, Mass.). The mobile phase consisted of 20:80acetonitrile:sodium acetate buffer (0.05 M, pH 4.0). The mobile phaseflow rate was 1.5 mL/min and the detection wavelength was 290 nm. 10 μLsamples were injected directly into the system using an auto-sampler.The method was validated in buffer and the coefficient of variation ofthe method was less than 2%. The HPLC standard curve was linear andfollowed the equation A=12.522C−87.465, where A is the absorbance and Cis the MTZ concentration in μg/mL. The limit of quantitation was 25ng/mL. All solvents used were HPLC grade.

A dialysate of 0.005N NaOH was perfused through microdialysis probeswith different continuous flow rates (q=3, 5, 7, 10, 15, 20, 30, 40, 50,and 60 μL/min). The probes were immersed in an MTZ solution of knownconcentration. For each flow rate, samples were collected into 250 μLmicrotubes and analyzed by HPLC.

A dialysate of 0.005N NaOH was pumped into microdialysis probes, allowedto sit inside the probe for different resting times, and then flushedout. The sample volumes were chosen to exceed the volume occupied insidethe microdialysis probe window (3.14 μL) plus the outer tubing (1.22μL). Samples were collected for each resting time and analyzed by HPLC.Studies were performed varying the resting times, sample volumes, flowrates and probe lengths as follows:

t_(R): 5, 7, 10, 15, 20, 25, 30, 40, 50, and 60 seconds

Vs: 5, 7, and 10 μL

Q: 50, 60, 80, 100, 150, and 200 μL/min

For each set of parameters, the pump was programmed to repeat the aboveprocedures seven times. The last five repetitions were collected andcombined to make up the sample to be assayed.

EXAMPLE 13 In Vitro Probe Calibration

PMD was performed for each probe window. The length of the window wasmeasured, and it was subsequently immersed in MTZ solutions of variousknown concentrations. A sample volume of 5 μL and flow rate of 100μL/min were selected. Samples were collected for each donorconcentration and immediately analyzed by HPLC. Calibration studies wereperformed for various values of t_(R) (10 and 15 seconds) and probewindow lengths (nominally 5 and 10 cm, corresponding to window volumesof ˜1.57 and 3.14 μL, respectively). All experiments were done intriplicate, and the calibration plots were constructed by plotting C_(S)vs. C_(D) according to Equation (1).

In addition to the initial probe calibration, subsequent “three-point”probe calibrations were done when appropriate to verify that theoriginal calibration curves were still valid. These were done byrepeating the above calibration procedure using three donorconcentrations over the donor concentration range of interest andcomparing the results with the original calibration.

In addition, it is possible to calibrate the microdialysis probe withrespect to the volume of the probe window, V_(W). This was done bychoosing a constant resting time that is long enough (>5-10 minutes) sothat F_(R)˜1, keeping the flow rate constant, and varying the samplevolume V_(S). V_(W) can be obtained from the slope of a plot of F_(R)vs. 1/V_(S), according to Equation (24). Here, F_(RQ) is the F_(R) forcontinuous flow and was determined from CFMD experimental data.

EXAMPLE 14 In Vitro Cellular Drug Uptake Simulation

The medium outside the probe served as the donor in this case. A donorsolution of MTZ was made with a known initial concentration C₀ andvolume V. The MTZ concentration was reduced over time by adding fresh0.005N NaOH solution (with no MTZ) to the solution at a constant flowrate q (volume/time) while stirring, and simultaneously removing thestirred solution at the same flow rate, both in a continuous manner.Using this setup, the volume of the solution remained constant and theMTZ concentration declined over time in a first order manner, accordingto the equation

C = C₀exp (−kt) $k = \frac{q}{V}$ $t_{1/2} = \frac{0.693}{k}$where k is the first order rate constant, and t_(1/2) is theconcentration half life (time for the concentration to drop in half),which is constant for this setup. In this experiment, the Co was 54mcg/mL, V was 50 mL, and q was 4.5 mL/min, which gave values of k=0.09min⁻¹ and t_(1/2)=7.7 minutes. PMD was performed using a 10 cm probewith a sample volume of 5 μL, flow rate of Q=100 μL/min, and restingtime of 10 seconds. Samples (25 μL) were collected at the same times (0,1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 50 and 60 minutes) from both thedonor solution and the dialysate, and immediately analyzed using HPLC.Three point probe calibrations were performed before each experiment.

EXAMPLE 15 Measuring a Rapid First Order Decline in the External MediumConcentration

The medium outside the probe served as the donor in this case. A donorsolution of sodium warfarin in an aqueous buffer (pH=7.4) was made witha known initial concentration C₀ and volume V. The warfarinconcentration was reduced over time by adding fresh buffer solution(with no warfarin) to the solution at a constant flow rate q(volume/time) while stirring, and simultaneously removing the stirredsolution at the same flow rate, both in a continuous manner. Using thissetup, the volume of the solution remained constant and the warfarinconcentration declined over time in a first order manner, according tothe equation

C = C₀exp (−kt) $k = \frac{q}{V}$ $t_{1/2} = \frac{0.693}{k}$where k is the first order rate constant, and t_(1/2) is theconcentration half life (time for the concentration to drop in half),which is constant for this setup. In this experiment, the C₀ was 100mcg/mL, V was 50 mL. Various flow rates q were used, including 135mL/min (half life of 16 seconds) and 230 mL/min (half life of 9seconds). PMD was performed using two 10 cm probes with a sample volumeof 22 μL (11 μL per probe), flow rate of Q=165 μL/min, and resting timeof 3 seconds. PMD samples (22 μL) were collected every 7 seconds for twominutes and immediately analyzed using HPLC. Three point probecalibrations were performed before each experiment.

The concentration in the donor was obtained as follows. From a staticprobe calibration at a resting time of 3 seconds, the value of γ₁ forthe probe was obtained from Equation (69). Subsequently, PMD wasperformed and single samples (i.e., samples were not combined) werecollected every 7 seconds using a resting time of 3 seconds. Theempirical function given by Equation (70) was fit to the PMD data usingEquations (72)-(74). From that fit, the plot times were taken as thebeginning of each PMD sample and the corresponding donor concentrationswere calculated using the FR given by Equation (75).

EXAMPLE 16 Charcoal MTZ Adsorption Kinetics Study

The medium outside the probe served as the donor in this case. Acharcoal suspension was prepared at least 24 hours before use by adding0.6 g of activated charcoal (Sigma Chemical, St. Louis, Mo.) to 100 mLof 0.6% Dextran Solution (Birkmeier et al., 1995). A donor solution ofMTZ in 0.005N NaOH was made with an initial concentration of C₀=200μg/mL and volume of 48 mL. A probe was immersed in the solution, and PMDwas started before adding the charcoal using a dialysate that initiallycontained no MTZ. Samples were collected every 10 seconds, using aresting time of 8 seconds, a flow rate of 100 μL/min, and a samplevolume of 5 μL. After collecting at least six samples, 2 mL of thecharcoal suspension was quickly (in 1-2 seconds) mixed into 48 mL ofstirred MTZ solution, giving a final volume of 50 mL. The time at whichthe charcoal was added was taken as the start time (t=0) for the bindingexperiment. Samples were collected during 10 second intervals, and thestart times for the sampling intervals were 0, 0.2, 0.4, 0.7, 1, 2, 5,10 and 30 minutes. The flow rate was 100 μL/min, the rest time was 8seconds, and the sample volume was 5 μL. The samples were immediatelyanalyzed using HPLC. The study was done at 37° C., and a three-pointprobe calibration was performed before and after each experiment. Directsampling of the donor was also done at various times for comparison withthe PMD data. The direct donor samples were immediately filtered toremove the activated charcoal and analyzed using HPLC.

EXAMPLE 17 Serum Bovine Albumin (BSA) Warfarin Protein Binding KineticsStudy

The medium outside the probe served as the donor in this case. A 4% (byweight) aqueous solution of bovine serum albumin (BSA) was prepared atleast 2 hours before use. An aqueous donor solution of warfarin wasprepared separately. Two probes were immersed in the solution, and PMDwas done using both probes simultaneously. PMD was started before addingthe BSA solution, using a dialysate that initially contained nowarfarin. Samples were collected every 7 seconds, using a resting timeof 3 seconds, a flow rate of 165 μL/min, and a sample volume of 11 μLper probe, giving a total sample volume of 22 μL. After collectingenough samples to eliminate any residual drug (typically at least 6samples, as in this example), the BSA solution was quickly added to thewarfarin solution and mixed (over 1-2 seconds), giving a nominal totalwarfarin concentration of 100 mcg/mL. The time at which the BSA wasadded was taken as the start time (t=0) for the binding experiment.Various amounts of BSA were used to give a range of proteinconcentrations ranging from 4 to 20 mg/mL. Simultaneously, PMD sampleswere collected every 7 seconds for three minutes, then at 5, 10, 15, 20,30, 40, 50, and 60 minutes. Samples were immediately analyzed by HPLC.The study was done at 37° C., and a three point probe calibration wasperformed before each experiment.

EXAMPLE 18 Benzocaine Release from Self-Emulsifying MicroemulsionSystems

The medium outside the probe served as the receiver in this case. Beforethe PMD experiments, various anhydrous liquid systems were made, to beused to release benzocaine. In all cases, two surfactants, Tween 20 andCremophor EL (both from Sigma Chemical, St. Louis, Mo.), were blended ina 2:1 (weight:weight) ratio. Various amounts of peppermint oil wereadded (from 0 to 30% oil by weight) to make the anhydrous systemsBenzocaine (50 mg per gram of peppermint oil) was subsequently added toeach system, and stirring was done until all benzocaine was dissolvedand distributed within the oil and surfactant blend. These anhydroussystems were then diluted 1:25 in water, and used as the dialysate. Aprobe was immersed in 50 mL of water (which served as the receiver) thatinitially contained no drug. Since benzocaine is a weak base, various pHvalues were used, but the pH of the water and dialysate were the same inall cases. The dialysate (containing the microemulsions and acting asthe donor) was pumped into the probe window, allowed to remain at restfor various resting times (5-100 seconds), pumped out and collected, andsubsequently assayed for benzocaine content to determine the fraction-ofbenzocaine lost as a function of the total time in the probe window. Theflow rate Q was 100 μL/min and the sample volume was 5 μL. The study wasdone at 25° C., and a three point probe calibration was performed beforeeach experiment.

EXAMPLE 19 Dissolution of Ibuprofen

The medium outside the probe served as the donor in this case. Anaqueous solution of NaOH in water at pH=2.0 was prepared, and 50 mL ofthe solution was used as the external medium. The solution wasmaintained at 25° C. and stirred continuously using a magnetic stirrer.A microdialysis probe was place into the external medium 30 minutesbefore beginning the dissolution experiment. After 30 minutes, 100 mg ofibuprofen (which exceeded its solubility in water at that pH) was addedto the medium. PMD was started before adding the ibuprofen, using adialysate that initially contained no drug. Each PMD sample was taken bycollecting five pulses with no waiting between pulses, discarding thefirst two and combining the next three. Each pulse was done using aresting time of 10 seconds, a flow rate of 100 μL/min, and a samplevolume of 10 μL (per pulse), giving a total sample volume of 30 μL.Samples were collected beginning at 1, 2, 3, 5, 10, 20, 30, 45, 60, 90and 120 minutes and immediately analyzed using HPLC. Since thedissolution rate was slow, the concentration in the donor was foundusing Equation (1). A three point probe calibration was performed beforeeach experiment.

EXAMPLE 20 Enzyme Assay Procedure

The medium outside the probe serves as the donor in this case. Anappropriate concentration of an enzyme is to be prepared before use. Anaqueous donor solution of the drug is prepared separately. As an option,the concentration of the drug and/or enzyme can be adjusted to speed upor slow down the degradation rate of a drug in the presence of theenzyme. A microdialysis probe (or set of probes for fast reactions) isimmersed in the drug solution, and PMD is started before adding theenzyme, using a dialysate that initially contains no drug. Samples arecollected at appropriate times, using appropriate resting times, flowrates and sample volumes. The concentration in the donor for the freedrug that has not yet undergone degradation is determined using Equation(1) or the method of Example 15. These choices will depend on whetherthe degradation occurs quickly (say, greater than 5% degradation perminute) or more slowly. (Compare Examples 14 and 17, for instance.)Three point calibrations are performed before and after each experiment.

Results and Discussion

Sensitivity of PMD

In this study, a tubular probe was used. A schematic diagram is shown inFIG. 1, in which the permeable tubing (probe window) is represented bythe broken lines and the impermeable tubing is represented by the heavysolid lines. The length of the probe window is L and the inner radius isa, and its volume is given by V_(W)=πa² L . The centerline (r=0) isrepresented by the dashed line.

The sensitivity of the PMD method was compared to that of the CFMDmethod by comparing the F_(R) as a function of the sample collectionrate. For CFMD, the sample collection rate is the volume of samplecollected per time, and equals the continuous flow rate Q. For PMD, thesample collection rate is the sample volume divided by the time tocollect the sample, and can thought of as an “average flow rate” (Q),given by

$\begin{matrix}{\left\langle Q \right\rangle = \frac{V_{S}}{t_{S}}} & (76)\end{matrix}$

Here, t_(S) is the time required to collect the entire sample, which isthe sum of the time required to flush the sample volume plus the restingtime, and is given by

$\begin{matrix}{t_{S} = {t_{R} + \frac{V_{S}}{Q}}} & (77)\end{matrix}$where Q is the flow rate used for flushing in the PMD experiment. Forthe PMD experiments, (Q) was changed by varying the resting time, whilekeeping the sample volume and flow rate for flushing constant (V_(S)=5μL and Q=100 μL/min).

A comparison is shown in FIG. 2. There, the solid diamonds/solid linerepresents PMD data, and the open squares/dashed line represents CFMDdata. In all cases, the temperature was 37° C., the probe window was11.24 cm in length and had a nominal inner radius of 100 μ, the flowrate for flushing 100 μL/min, and the sample volume V_(S) was 5 μL. FIG.2 shows that, at similar sample collection rates, the PMD and CFMD datadisplayed similar values of F_(R), which decreased with increasing flowrates for both methods due to shorter exposure times.

The minimum measurable donor concentration was estimated by dividing theassay limit of quantitation (the lowest concentration that can beassayed by the HPLC with acceptable error) by the F_(R). In this study,the HPLC limit for MTZ was ˜25 ng/mL. For a 10 second resting time and10 cm probe window, F_(R)˜0.2 and the minimum measurable C_(D) was ˜125ng/mL. For the same system and a 30 second t_(R), F_(R)˜0.6 and theminimum measurable C_(D) was ˜40 ng/mL. Thus, the sensitivity of the PMDmethod can be effectively adjusted by varying the resting time.

While PMD and CFMD show comparable sensitivity in determining drugconcentrations, there are two important differences. First, if theconcentration of drug in the medium outside the probe does not change,then all parts of a CFMD sample are identical. On the other hand, in thecase of PMD, the continuous (flushed) portions are identical, but thepulsed (rested) portions are different if different resting times areused. In addition, while the pulsed portion is at rest, drug transportoccurs by diffusion alone. Thus, a body of diffusion equations becomesavailable in PMD that allows the determination of properties such asdiffusion coefficients, probe window permeabilities, etc. Since there isno passive diffusion phase in CFMD, these analyses are not applicable tothat method.

The second difference occurs because the dialysate flow rate is constantand continuous in CFMD, so the sample is collected continuously over thesample interval (which is typically long). If the donor is outside theprobe and the drug concentration changes significantly during thesampling interval, it is not feasible to assign an exact concentrationto an exact time using CFMD. PMD is much better suited for this purpose,especially if the flushing is done using high flow rates, because almostall of the drug is accumulated in the sample during the resting time. Ifthat resting time is short, PMD gives a “snapshot” of the system, and itis possible assign a donor concentration to a specific time.

Calibration Plots

To correlate the concentration between the sample and donor, calibrationplots were made by plotting the C_(S) vs. C_(D). In CFMD, this is donein vitro by comparing dialysate sample concentrations with those ofdirect sampling of the donor, and the assumption is made that therelation between C_(S) and C_(D) does not change during the experiment.In this study, similar calibration plots were made using data obtainedby PMD. It was observed that calibration plots were linear (R²>0.99) inall cases. Some data is shown in FIGS. 3 and 4. FIG. 3 shows calibrationcurves for two different probes. There, the dashed line (triangles)represents data generated using a probe window of 11.24 cm length, andthe solid line (circles) represents data generated using a probe windowof 11.04 cm length. In both cases, the temperature was 37° C., thenominal inner radius of the probe window was 100 μ, the resting time was10 seconds, the sample volume was 5 μL and the flow rate was 100 μL/min.Both calibration curves were linear (R²>0.99). The dashed line isdescribed by the equation C_(S)=0.207C_(D)+21.53, and the solid line isdescribed by C_(S)=0.170C_(D)+30.17. (The concentrations are in units ofμg/mL.) Since the intercepts were close to the origin, the fractionrecovered F_(R) for a given probe was taken as being equal to the slopeof the C_(S) vs. C_(D) plot. This shows that there may be some variationbetween microdialysis probes, so each must be calibrated individually.

The observed F_(R) is affected by the exposure time of the sample,increasing with longer exposure. This is shown in FIG. 4, in which thesolid line (diamonds) represents data generated using resting times of10 seconds, and the dashed line (squares) represents data generatedusing resting times of 15 seconds. In both cases, the temperature was37° C., the length of the probe window was 11.24 cm, the inner radius ofthe probe window was 100 μ, the sample volume was 5 μL and the flow ratewas 100 μL/min. Both calibration curves were linear (R²>0.99). For the10 second resting time data, the data fit the line C_(S)=0.207C_(D)+20.97 (F_(R)=0.207). For the 15 second resting_(time) data, thedata fit the line C_(S)=0.281 C_(D)+19.11 (F_(R)=0.281). Theconcentrations are in units of μg/mL. Thus, for the probes shown in FIG.4, increasing the exposure time from 10 seconds to 20 seconds increasesthe F_(R) from 0.207 to 0.281. This is because longer exposure of thedialysate to the donor solution allows more of the drug to diffuse intothe dialysate. This effect is also illustrated in FIG. 5, which shows aplot of F_(R) vs. t_(P) for the 11.24 cm probe from FIG. 4. In FIG. 5,the squares represent experimental data points, and the line representsthe best fit to the data using Equations (21)-(24).

The F_(R) decreased with increasing sample volume and increased with thevolume of the probe window in a manner that was also consistent withEquation (24). A plot of F_(R) vs. 1 /V_(S) is shown in FIG. 6. There,the temperature was 37° C., the length of the probe window was 5 cm, thenominal inner radius of the probe window was 100 μ, and the flow ratewas 100 μL/min. The sample volume was varied, and the solid line is thelinear best fit curve through the data points. The line has a slope of2.71 and an intercept of 0.06 (R²>0.99), corresponding to V_(W)=2.9 μL.This value of V_(W) differs somewhat from the value calculated based onthe manufacturer's data for the probe radius. (Using the nominal radiusof 100 μ, and measured window length of 5 cm gives a nominal windowvolume of 1.5 μL.). Using optical microscopy, it was verified that theprobe diameter was significantly larger the nominal value, but theaccuracy of optical microscopy is still inferior to the procedure usedhere.) In addition, the intercept (0.06) is in agreement with the F_(R)obtained from CFMD experiments using the same flow rate. This is to beexpected because the intercept occurs when 1/V_(S)→0, which physicallycorresponds to CFMD.

FIG. 7 shows a plot of ln(1−F_(RP)) vs. t_(P), as given by Equation (30)or (35), which is linear as expected. MTZ was used as the referencedrug, which has a value of 4×10⁻⁶ cm²/s at 37° C., and F_(R) was foundfrom F_(R) using Equation (24), using a window volume of 3.6 μL. Thetemperature was 37° C., the probe window length was 11.24 cm, the probewindow inner radius was 100 the sample volume was 5μL and the flow ratewas 100 μL/min. The line is linear (slope=−0.0187 s⁻¹, intercept=−0.007,R²>0.99). From these results, the permeability of the probe window wallwas P=9.4×10⁻⁵ cm/s and 2=0.23, which was in the expected range for thisprobe.

Time Resolution and Simulated Uptake Studies

The ability to detect relatively rapid changes in the donorconcentration can be an important consideration for systems in which theconcentration changes rapidly, such as might occur with in vitrocellular uptake or binding studies. Since the CFMD method typicallyrequires several minutes for sample collection, it is not possible toassign accurate times (either exact or to within short time intervals)to the dialysate sample concentrations. This is one of the primaryreasons for inventing the PMD method, which can assign concentrations toshort and specific time intervals, which correspond to the samplinginterval time t_(S) defined in Equation (77).

For systems in which the donor concentration is changing, plots ofconcentration (or some function of the concentration) vs. time areusually the preferred way to view or analyze the experimental data. Forthese PMD experiments, the concentration being is determined from thedialysate sample concentration, which can be used as is or used tocalculate the concentration in the medium outside the probe usingEquation (1) (depending on the experiment being done). The time t atwhich that concentration is plotted is taken as

Plot  time  t = [time  after  the  start  of  the  experiment  when  a  particular  PMD  sampling  begins] +   [half  the  time  to  take  the  PMD  sample]which can be calculated as

$\begin{matrix}{t = {t_{plot} = {t_{{begin}\mspace{14mu}{sample}} + \frac{t_{S}}{2}}}} & (78)\end{matrix}$

When the donor concentration changes rapidly, a more accurate procedureis used to determine the donor concentration and plot time. This is doneusing the analysis leading to Equations (72)-(75) and doing thenonlinear regressions to determine the fitting parameters used in anappropriate fitting function, such as that given by Equation (71). Thisshould be used when the change in the donor concentration between thetime an individual PMD sample is started and complete is significant.(For instance, more than 5% loss, although this could be greater orsmaller, depending on the accuracy needed in an experiment. For proteinbinding experiment, an error greater than 5% would be undesirable, forexample.)

As mentioned above, one of the features that makes PMD well suited forsampling changing concentrations is that the time during which thedialysate accumulates or loses a compound by diffusion is well defined,so specific concentrations can be assigned to specific times and/or timeintervals. Another advantage is that the time interval t_(S) can be veryshort (as short as four or five seconds), which is very important whensampling concentrations that change, but is not possible with othertypes of experimental setups. The ability to sample in short intervalsoccurs because the radii of the probes are very small, which has twoimportant implications.

-   -   The diffusing molecules do not have to travel very far (relative        to other methods) before a significant portion of the dialysate        volume gains or loses molecules. Thus, in a given time interval,        a larger fraction of the dialysate is affected by drug        diffusion.    -   The surface/volume ratio of the probe is very large (relative to        other methods). This is relevant because the rate at which        molecules diffuse across the probe wall is proportional to its        area, while the change in dialysate concentration is inversely        proportional to its volume. Thus, in a given time interval,        larger surface/volume ratios lead to larger concentration        changes in the dialysate.    -   The combined effect is that PMD can measure concentrations and        concentration changes (inside or outside the probe) by taking        samples over short intervals t_(S). This was tested in several        experiments, as discussed below.

A first order uptake was simulated by adding 4.5 mL/min to 50 mL ofstirred MTZ solution in NaOH, and simultaneously removing 4.5 mL/min ofthe mixture. This maintained a constant donor solution volume, but theMTZ concentration declined according to the equation C=C₀ exp(−kt).Based on the pump rates, the calculated value of the rate constant wask=0.090 min⁻¹ (half-life of 7.7 hours). FIG. 8 shows the results forthat experiment using a temperature of 37° C., probe window length of11.24 cm, nominal inner radius of the probe window of 100 sample volumeof 5 μL, and the flow rate of 100 μL/min. The value found from directsampling of the donor solution (dashed line) was k=0.088 min⁻¹, and thevalue found from the PMD data (solid line) was k=0.089 min⁻¹. Theintercept from the direct sampling data=10.887 and the intercept fromthe PMD data=9.150. Theoretically, the difference between the interceptsof the In C vs. t plots for the PMD and direct donor sampling datashould be equal to ln (F_(R)). From the data shown in FIG. 8, theintercepts for PMD and direct sampling differ by −1.737, giving anF_(R)=0.176. This value is nearly identical to the of 0.178 obtainedfrom the calibration plot for this PMD setup.

In the second experiment, a more rapid first order decline in donorconcentration was brought about. This was done for the drug sodiumwarfarin in an aqueous buffer by adding fresh buffer to 50 mL of stirredwarfarin solution at various flow rates and simultaneously removing thestirred mixture at the same flow rate. The temperature was 37° C., twoprobes were used simultaneously (window volume was 5 μL per probe), thesample volume was 22 μL (11 μL per probe), the resting time was 3seconds and the flow rate was 165 μL/min. The same equations used forthe MTZ experiment apply, but the pump rates ranged up to 230 mL/min.Based on the pump rates, the calculated values of the rate constant wereas high as k=0.077 sec⁻¹. Donor concentrations calculated using PMD andthe model leading to Equations (72)-(75) were in excellent agreement,using the fitting function given by Equation (71). Data for the 16second half life experiments, in which the pumping rates were chosen tomimic a first order rate constant of k=0.043 sec⁻¹, are shown in FIG. 9.There, the open circles represent the PMD concentrations, the filledtriangles represent the donor concentration plotted at appropriatetimes, as calculated from the PMD sample data. The solid line representsthe theoretical value of the donor concentration that was calculatedusing the theoretical rate constant. It can be seen that the calculatedand theoretical donor concentrations are in excellent agreement. Sincethe decline was designed to be first order, a plot of the natural log ofthe calculated (from PMD data) and theoretical donor concentrations fromthe first order rate constant) vs. time is shown in FIG. 10. There, thefilled triangles represent the donor concentration plotted atappropriate times, as calculated from the PMD sample data. The solidline represents the theoretical value of the donor concentration thatwas calculated using the theoretical rate constant. FIG. 10 shows thatthe rate constant k for the calculated and theoretical donorconcentrations are in excellent agreement (k=0.044 sec⁻¹ for the declinecalculated from PMD data vs. k=0.043 sec⁻¹ for the theoretical valuecalculated from the pumping rates).

In a third experiment, activated charcoal was added to an MTZ solutionand the donor concentrations were simultaneously sampled using PMD anddirect donor sampling. In this system, the donor concentration droppedrapidly over the first minute, and declined slowly after that. Theresults are shown in FIG. 11 for the first five minutes of theexperiment (after which time the concentrations did not change). In thatexperiment, the temperature was 37° C., the length of the probe windowwas 11.24 cm, the nominal inner radius was 100 μ, the sample volume was5 μL and the flow rate was 100 μL/min. In the figure, the diamondsrepresent data obtained using PMD, and the large open circles representdata obtained from direct donor sampling. The data obtained using PMDand direct donor sampling were in excellent agreement, and indicatedthat the free MTZ concentration fell from 5.4 to 2.8 μg/mL over thefirst minute after the addition of the activated charcoal. (It should benoted that direct donor samples taken before the first minute were veryinconsistent. This is because adsorption was still going on while thecharcoal and free drug were being separated.) In addition, the PMDmethod was able to generate six concentration measurements over thefirst minute. Thus, PMD was able to characterize the concentration vs.time profile, even during the first minute of the experiment (when theconcentration declined by nearly 50%). As a rough check for consistencyof the PMD data, the rate of change of C_(D) was plotted as a functionof time. This plot was smooth, continuous and the rate monotonicallyapproached zero. (While this does not imply anything about the physicalprocess that governs the adsorption kinetics, all of thesecharacteristics should be expected from experimental data.)

In an analogous experiment using proteins, a solution of BSA (bovineserum albumin) was added to an aqueous solution of sodium warfarin inphosphate buffer, and the donor concentrations were sampled using PMD.The temperature was 37° C., the pH was 7.4, the concentration of the BSAsolution before mixing with the warfarin solution was 40 mg/mL. Aftermixing the warfarin and BSA solutions, the total warfarin concentrationwas 102 mcg/mL (0.31 mM) and the total BSA concentration was 16 mg/mL.The results are shown in FIG. 12, in which the points represent theexperimental warfarin concentrations and the solid line represents thebest fit curve using a postulated binding model. It was seen that thedonor warfarin concentration dropped rapidly over the first minute, anddeclined slowly after that, and reached equilibrium after a few minutes.The results are shown only for the first 90 seconds of the experiment,after which time the concentrations did not change. The PMD method wasable to generate eight concentration measurements over the first minuteand was able to characterize the concentration vs. time profile for theentire course of the binding process.

In a different experiment, the release of benzocaine from variousmicroemulsion systems was studied. In these experiments, the donorliquid was the dialysate and the receiver was the medium outside of theprobe. Thus, PMD was used to measure the amount of drug lost by thedialysate, and the fraction remaining R_(F) was calculated. Themicroemulsion systems used here contained Tween 20:CremophorEL:peppermint oil mixtures, which were diluted in water after mixing ina 1:25 weight ratio. For these systems, the release of the drug wasrelatively rapid (losing as much as 90% of the benzocaine when occupyingthe probe window for one minute). FIG. 13 shows the log of the fractionremaining vs. time for the release of benzocaine from a microemulsion at25° C., in which the surfactant was a 2:1 (weight/weight) blend of Tween20 plus Cremophor EL, and the surfactant:peppermint oil weight ratio was90:10 (before adding water). The triangles represent the experimentaldata, the line represents the best fit line. (On the vertical axis, thenotation f_(rem) is used to denote the R_(F), which is the fraction ofthe total drug loaded remaining in the dialysate.)

I claim:
 1. A method for accurately determining the rate of release ofan agent dissolved in emulsion and/or microemulsion droplets,comprising: a) providing a probe comprising a section of relativelyhighly permeable membrane relative to any materials to which themembrane is attached for support and positioned between an inlet to asource of emulsion and an outlet to a receptacle, and through whichmembrane particles are to be transferred; b) putting said probe incontact with a medium into which the particles are to be transferred bydiffusion; c) perfusing a known quantity (V_(W)) of dialysate containingsaid emulsion into the relatively highly permeable section of the probe;d) allowing said known quantity of emulsion to remain stationary for aresting time (t_(R)) sufficient to permit at least some of said emulsionparticles to diffuse into the said medium; e) flushing out said knownquantity of emulsion with a single pulse of a known volume (V_(S)) ofemulsion into said receptacle at a flow rate sufficiently high that lessthan about 10% of said agent transfers out of the dialysate into saidmedium; f) determining the concentration of said agent remaining in saidknown volume (V_(S)); g) repeating steps (c) through (f) with at leastone different (t_(R)); h) plotting the results of steps (f) and (g) todetermine how said concentration changes with (t_(R)); i) taking theslope of the plot of step (h), which is the rate of release of saidagent from the emulsion.
 2. A method for accurately determining the rateat which a diffusible agent is dissolved or released from an agentcontaining said diffusible agent, comprising: a) providing a probecomprising a section of relatively highly permeable membrane relative toany materials to which the membrane is attached for support andpositioned between an inlet to a source of dialysate and an outlet to areceptacle, and through which membrane a diffusible agent is to betransferred; b) determining the value of γ₁ for the probe using a chosensample volume, flow rate and resting time; c) putting said probe incontact with said medium which may or may not contain the agent to betransferred by diffusion; d) to said medium, adding the agent containingsaid diffusible agent; e) perfusing a known quantity of a dialysate intothe relatively highly permeable section of the probe at the same flowrate Q used to calculate γ₁; f) allowing said known quantity ofdialysate to remain stationary for the same resting time t_(R) used tocalculate γ₁; g) flushing out said known quantity of dialysate with asingle pulse to collect a sample of dialysate, of the same volume asthat chosen in step (b), of a known volume V_(S), dialysate into saidreceptacle at the same flow rate used in step (d), above; h) determiningthe concentration of said diffusible agent in said dialysate; i)repeating steps (e) through (h) with the same flow rate, sample volume,and resting time; j) determining the fractional recovery at thebeginning of a sample interval by calculating F_(R) ⁰; k) calculatingthe concentration in the medium at the beginning of the sample intervalas C_(D)=C_(S)/F_(R) ⁰.
 3. A method of claim 2 in which said agent is asolid or mixture of solids.
 4. A method of claim 2 in which said agentis a liquid.